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. 2024 Jan 29:13:e93517.
doi: 10.7554/eLife.93517.

The tRNA thiolation-mediated translational control is essential for plant immunity

Xueao Zheng #  1   2   3   4   5   6 , Hanchen Chen #  1   3   4   5   6 , Zhiping Deng  7 , Yujing Wu  1   3   4   5   6 , Linlin Zhong  8 , Chong Wu  1   3   4   5   6 , Xiaodan Yu  1   3   4   5   6 , Qiansi Chen  2 , Shunping Yan  1   3   4   5   6
Affiliations

The tRNA thiolation-mediated translational control is essential for plant immunity

Xueao Zheng et al. Elife. .

Abstract

Plants have evolved sophisticated mechanisms to regulate gene expression to activate immune responses against pathogen infections. However, how the translation system contributes to plant immunity is largely unknown. The evolutionarily conserved thiolation modification of transfer RNA (tRNA) ensures efficient decoding during translation. Here, we show that tRNA thiolation is required for plant immunity in Arabidopsis. We identify a cgb mutant that is hyper-susceptible to the pathogen Pseudomonas syringae. CGB encodes ROL5, a homolog of yeast NCS6 required for tRNA thiolation. ROL5 physically interacts with CTU2, a homolog of yeast NCS2. Mutations in either ROL5 or CTU2 result in loss of tRNA thiolation. Further analyses reveal that both transcriptome and proteome reprogramming during immune responses are compromised in cgb. Notably, the translation of salicylic acid receptor NPR1 is reduced in cgb, resulting in compromised salicylic acid signaling. Our study not only reveals a regulatory mechanism for plant immunity but also uncovers an additional biological function of tRNA thiolation.

Keywords: A. thaliana; Arabidopsis; NPR1; plant biology; plant immunity; tRNA thiolation; translation.

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Conflict of interest statement

XZ, HC, ZD, YW, LZ, CW, XY, QC, SY No competing interests declared

Figures

Figure 1.
Figure 1.. The rol5 mutants are more susceptible to the bacterial pathogen Psm ES4326 than wild-type (WT).
(A) Pictures of Arabidopsis 3 days after infection. The arrows indicate the leaves inoculated with Psm ES4326 (OD600=0.0002). cgb and rol5-c are mutants defective in ROL5. COM, the complementation line of cgb. npr1-1 serves as a positive control. Bar = 1 cm. (B) The growth of Psm ES4326. CFU, colony-forming unit. Error bars represent 95% confidence intervals (n=7). Statistical significance was determined by two-tailed Student’s t-test. ***, p<0.001; ns, not significant. (C) A schematic diagram showing the site of the T-DNA insertion in cgb and the deleted nucleotides in rol5-c. (D) The genotyping results using the primers indicated in C. (E) The transcript of ROL5 is not detectable in cgb. UBQ5 serves as an internal reference gene.
Figure 2.
Figure 2.. ROL5 interacts with CTU2.
(A) A schematic diagram showing the function of ROL5 and CTU2. The ROL5 homolog NCS6 and the CTU2 homolog NCS2 form a complex to catalyze the mcm5s2U modification at wobble nucleotide of tRNA-Lys (UUU), tRNA-Gln (UUC), and tRNA-Glu (UUG), which pair with the AAA, GAA, and CAA codons in mRNA, respectively. (B) Yeast two-hybrid assays. The growth of yeast cells on the SD-Trp/Leu/His medium indicates interaction. BD, binding domain. AD, activation domain. (C) Split luciferase assays. The indicated proteins were fused to either the C- or N-terminal half of luciferase (cLUC or nLUC) and were transiently expressed in N. benthamiana. The luminesce detected by a CCD camera reports interaction. (D) Co-immunoprecipitation (CoIP) assays. CTU2-GFP and/or ROL5-FLAG fusion proteins were expressed in N. benthamiana. The protein samples were precipitated by GFP-Trap, followed by western blotting using anti-GFP or anti-FLAG antibodies. (E) GST pull‐down assays. The recombinant GST or GST-CTU2 proteins coupled with glutathione beads were used to pull down His-ROL5, followed by western blotting using anti-His or anti-GST antibodies.
Figure 3.
Figure 3.. ROL5 and CTU2 are required for mcm5s2U modification and plant immunity.
(A and B) The rol5-c and ctu2-1 mutants are more susceptible to the bacterial pathogen Psm ES4326 than wild-type (WT). (A) Pictures of Arabidopsis plants 3 days after infection. Arrows indicate the leaves inoculated with Psm ES4326. Bar = 1 cm. (B) The growth of Psm ES4326. CFU, colony-forming unit. Error bars represent 95% confidence intervals (n=6). Statistical significance was determined by two-tailed Student’s t-test. ***, p<0.001. (C) The rol5-c and ctu2-1 mutants lack the mcm5s2U modification. The levels of U, cm5U, mcm5U, and mcm5s2U were quantified through high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) analyses. The intensity and the retention time of each nucleotide are shown. The structure of each nucleotide and the catalyzing enzymes are shown on the right.
Figure 4.
Figure 4.. The transcriptome and proteome reprogramming are compromised in cgb.
(A and B) The percentage and the number of the differentially expressed genes (DEGs, p-value <0.05, |Log2Foldchange|>Log21.5, (A)) and the differentially expressed proteins (DEPs, p-value <0.05, |Log2Foldchange|>Log21.2, (B)) after Psm infection in the cgb mutant and the complementation line (COM). Down, down-regulated. Up, up-regulated. Nc, no change. (C and D) The percentage and the number of the attenuated genes (C) and proteins (D) in cgb among the up-regulated DEGs and DEPs in COM. (E and F) Gene Ontology (GO) analysis of the attenuated genes (E) or proteins (E) in cgb. The top 15 significantly enriched GO terms are shown.
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. Principal component analysis (PCA) of the transcriptome (A) and proteome samples (B).
Figure 5.
Figure 5.. The translation of immune-related proteins is compromised in cgb.
(A) Venn diagram analysis of attenuated genes and proteins. (B) Gene Ontology (GO) analysis of the 261 attenuated proteins. The top 6 significantly enriched GO terms are shown. (C) Western blot analysis of NPR1 protein levels. The 7-day-old seedlings grown on 1/2 MS medium were treated with buffer (10 mM MgCl2, pH 7.5, Mock) or Psm ES4326 (OD600=0.2) for 48 hr. (D) Polysome profiling results. Abs, the absorbance of sucrose gradient at 254 nm. The numbers on the X-axis indicate the polysomal fractions subjected to qPCR analyses. (E) The qPCR analyses. The relative mRNA level of NPR1 in different fractions or in total mRNA was normalized against UBQ5. The ratio between the relative mRNA levels in each fraction and in total mRNA was shown (n=3). Statistical significance was determined by two-tailed Student’s t-test. **, p<0.01; ***, p<0.001; ns, not significant. (F) The heatmap showing the expression changes of salicylic acid (SA)-responsive genes after pathogen infection.
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. Analyses of NPR1 transcript levels in cgb and COM.
The 7-day-old seedlings grown on 1/2 MS medium were treated with buffer (10 mM MgCl2, pH 7.5, Mock) or Psm ES4326 (OD600=0.2) for 48 hr. The relative mRNA level of NPR1 was normalized against UBQ5. Error bars represent 95% confidence intervals (n=3). Statistical significance was determined by two-tailed Student’s t-test. ns, not significant.
Figure 5—figure supplement 2.
Figure 5—figure supplement 2.. The salicylic acid (SA)-mediated protection assay.
The Arabidopsis plants were treated with (+) or without (-) 600 μM benzothiadiazole (BTH) for 24 hr before infection. The growth of Psm ES4326 was shown. CFU, colony-forming unit. Error bars represent 95% confidence intervals (n=7). Statistical significance was determined by two-tailed Student’s t-test. ***, p<0.001; ns, not significant.
Figure 5—figure supplement 3.
Figure 5—figure supplement 3.. The genetic relationship between NPR1 and CGB.
The Arabidopsis plants were infected with Psm ES4326 and the growth of Psm ES4326 was shown. CFU, colony-forming unit. Error bars represent 95% confidence intervals (n=7). Statistical significance was determined by two-tailed Student’s t-test. **, p<0.01.
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References

    1. Agris PF, Eruysal ER, Narendran A, Väre VYP, Vangaveti S, Ranganathan SV. Celebrating wobble decoding: half a century and still much is new. RNA Biology. 2018;15:537–553. doi: 10.1080/15476286.2017.1356562. - DOI - PMC - PubMed
    1. Canet JV, Dobón A, Roig A, Tornero P. Structure-function analysis of npr1 alleles in arabidopsis reveals a role for its paralogs in the perception of salicylic acid. Plant, Cell & Environment. 2010;33:1911–1922. doi: 10.1111/j.1365-3040.2010.02194.x. - DOI - PubMed
    1. Cao H, Glazebrook J, Clarke JD, Volko S, Dong X. The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell. 1997;88:57–63. doi: 10.1016/s0092-8674(00)81858-9. - DOI - PubMed
    1. Chen H, Zou Y, Shang Y, Lin H, Wang Y, Cai R, Tang X, Zhou JM. Firefly luciferase complementation imaging assay for protein-protein interactions in plants. Plant Physiology. 2008;146:368–376. doi: 10.1104/pp.107.111740. - DOI - PMC - PubMed
    1. Chen H, He C, Wang C, Wang X, Ruan F, Yan J, Yin P, Wang Y, Yan S. RAD51 supports DMC1 by inhibiting the SMC5/6 complex during meiosis. The Plant Cell. 2021;33:2869–2882. doi: 10.1093/plcell/koab136. - DOI - PMC - PubMed

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