The tRNA thiolation-mediated translational control is essential for plant immunity
- Hubei Hongshan Laboratory, China
- Zhengzhou Tobacco Research Institute of CNTC, China
- College of Life Science and Technology, Huazhong Agricultural University, China
- Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, China
- Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, China
- Shenzhen Institute of Nutrition and Health, Huazhong Agricultural University, China
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, China
- Key Laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, China
Figures
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.
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Figure 1—source data 1
Source data related to Figure 1B.
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Figure 1—source data 2
Source data related to Figure 1D.
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Figure 1—source data 3
Source data related to Figure 1E.
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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.
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Figure 2—source data 1
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Figure 2—source data 2
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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.
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Figure 3—source data 1
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Figure 3—source data 2
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Figure 4 with 1 supplement
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.
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Figure 4—source data 1
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Figure 4—source data 2
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Figure 4—source data 3
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Figure 4—source data 4
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Figure 4—source data 5
Source data related to Figure 4E.
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Figure 4—source data 6
Source data related to Figure 4F.
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Figure 4—figure supplement 1
Principal component analysis (PCA) of the transcriptome (A) and proteome samples (B).
Figure 5 with 3 supplements
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.
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Figure 5—source data 1
Source data related to Figure 5A.
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Figure 5—source data 2
Source data related to Figure 5B.
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Figure 5—source data 3
Source data related to Figure 5C.
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Figure 5—source data 4
Source data related to Figure 5D.
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Figure 5—source data 5
Source data related to Figure 5E.
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Figure 5—source data 6
Source data related to Figure 5F.
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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
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
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.
Author response image 1
Author response image 2
Author response image 3
Author response image 4
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Arabidopsis thaliana) | ROL5 | TAIR | AT2G44270 | |
| Gene (Arabidopsis thaliana) | CTU2 | TAIR | AT4G35910 | |
| Genetic reagent (Arabidopsis thaliana) | cgb | This paper | It contains a T-DNA insertion in the fourth exon of ROL5 and is hypersusceptible to pathogen. | |
| Genetic reagent (Arabidopsis thaliana) | COM | This paper | It contains the coding sequence of ROL5 driven by 35S promoter in cgb. | |
| Genetic reagent (Arabidopsis thaliana) | rol5-c | This paper | The mutant was generated using CRISPR-Cas9 system. It contains a 2-bp deletion in the first exon of ROL5. | |
| Genetic reagent (Arabidopsis thaliana) | ctu2-1 | ABRC | SALK_032692 | |
| Genetic reagent (Arabidopsis thaliana) | npr1-1 | Cao et al., 1997 | ||
| Strain, strain Background (Escherichia coli) | BL21 | TransGen | Cat # CD901-02 | Electrocompetent cells |
| Strain, strain background (Escherichia coli) | DH5α | TransGen | Cat # CD201-01 | Electrocompetent cells |
| Strain, strain background (Agrobacterium tumefaciens) | GV3101 | Sangon | Cat # B528430 | Electrocompetent cells |
| Strain, strain background (Saccharomyces cerevisiae) | AH109 | Clontech | Cat # 630489 | Electrocompetent cells |
| Strain, strain background (Pseudomonas syringae pv. Maculicola) | Psm 4326 | Durrant et al., 2007 | ES4326 | |
| Antibody | Anti-NPR1 (Rabbit polyclonal) | From Dr. Li Yang | WB(1:3000) | |
| Antibody | Anti-His (Mouse monoclonal) | Abclonal | Cat # AE003 | WB(1:5000) |
| Antibody | Anti-GST (Mouse monoclonal) | Abclonal | Cat # AE001 | WB(1:5000) |
| Antibody | Anti-FLAG (Mouse monoclonal) | Promoter | WB(1:5000) | |
| Antibody | Anti-GFP (Mouse monoclonal) | Promoter | WB(1:5000) | |
| Other | GFP-Trap | chromotek | Cat # gtma | |
| Other | Hypersil GOLD | Thermo Fisher | Cat # 25005-254630 |
Appendix 1—table 1
The primers used in this study.
| Name | Sequence（5'–3'） | Application |
|---|---|---|
| ROL5-F1 | ACATTACAATTACATTTACAATTACATGGAGGCCAAGAACAAGAA | For complementation |
| ROL5-R1 | GGGTCTTAATTAACTCTCTAGATTAGAAATCCAGAGATCCACAT | |
| ROL5-F2 | CGGAATTC ATGGAGGCCAAGAACAAGA | For Y2H |
| ROL5-R2 | CGGGATCC TTAGAAATCCAGAGATCCAC | |
| CTU2-F1 | CGGAATTC ATGGCTTGTAATTCCTCAG | |
| CTU2-R1 | CGGGATCC TTAGACAACCTCTTCATCGT | |
| ROL5-F3 | GGGGTACCATGGAGGCCAAGAACAAGA | For split luc |
| ROL5-R3 | GCGTCGACGAAATCCAGAGATCCAC | |
| CTU2-F2 | GGGGTACCATGGCTTGTAATTCCTCAG | |
| CTU2-R2 | GCGTCGACTTAGACAACCTCTTCATCGT | |
| GUS-F | acgcgtcccggggcggtaccATGGTAGATCTGAGGGTAAA | |
| GUS-R | cgaaagctctgcaggtcgacCTATTGTTTGCCTCCCTGCTG | |
| ROL5-F0 | TGACTGCTCCCTACCTGTCGAGTTTTAGAGCTAGAAATAGC | For CRISPR mutant of ROL5 |
| ROL5-R0 | AACGAGACGTCCCGTCCTCAAACAATCTCTTAGTCGACTCTAC | |
| ROL5-BsF | ATATATGGTCTCGATTGACTGCTCCCTACCTGTCGAGTT | |
| ROL5-BsR | ATTATTGGTCTCGAAACGAGACGTCCCGTCCTCAAACAA | |
| ROL5-F4 | TTGAAAGGTTTACATCTTGGAAT | For sequencing of target sites |
| ROL5-R4 | AAAGGTGATTGCTTAGATTCTGATT | |
| ROL5-F5 | CTCAAAAACCTCATAAAAGCACTCT | |
| ROL5-R5 | AACTGCGTCACTGTCTTTACTCT | |
| ROL5-F6 | TTAAGAAGGAGATATACCATGGGCATGGAGGCCAAGAACAAGA | For protein expression |
| ROL5-R6 | GAGTGCGGCCGCAAGCTTTTAGAAATCCAGAGATCCAC | |
| CTU2-F3 | TTCCAGGGGCCCCTGGGATCCATGGCTTGTAATTCCTCAG | |
| CTU2-R3 | AGTCACGATGCGGCCGCTCGAGTTAGACAACCTCTTCATCGT | |
| ROL5-F7 | CAATTACATTTACAATTACATGGAGGCCAAGAACAAGA | For co-immunoprecipitation |
| ROL5-R7 | GGGTCTTAATTAACTCTCTAGATTTGTCATCATCGTCTTTG | |
| CTU2-F4 | CAATTACATTTACAATTACATGGCTTGTAATTCCTCAGG | |
| CTU2-R4 | GGGTCTTAATTAACTCTCTAGATTACTTGTACAGCTCGTCCA | |
| cgb-LP | GTATGAGAAGTGATTGAGTATGTG | For genotyping |
| cgb-RP | TCGATGTGCACCTACTTAATCTAC | |
| cgb-RB | CTAATGAGTGAGCTAACTCAC | |
| ctu2-LP | TCACATTGCATTGAATCATCC | For genotyping |
| ctu2-RP | TCAAATTTAGCACATGGGACC | |
| ROL5-F1 | GGAGCTGCGTTATTGAAAGTAG | For qPCR |
| ROL5-R1 | CCACGATATGCATTAGGAGAGT | |
| UBQ5-F1 | GAAGATCCAAGACAAGGAAGGA | |
| UBQ5-R1 | CTTCTTCCTCTTCTTAGCACCA | |
| NPR1-P1 | ATGATTTCTACAGCGACGCTAA | |
| NPR1-P2 | GACTTCGTAATCCTTGGCAATC |
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The tRNA thiolation-mediated translational control is essential for plant immunity
eLife 13:e93517.
https://doi.org/10.7554/eLife.93517
