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 …
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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 …
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Figure 2—source data 1
Source data related to Figure 2D.
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Figure 2—source data 2
Source data related to Figure 2E.
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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 …
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Figure 3—source data 1
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Figure 3—source data 2
Source data related to Figure 3C.
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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, …
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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
Source data related to Figure 4C.
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Figure 4—source data 4
Source data related to Figure 4D.
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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 …
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Figure 5—source data 1
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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.
- https://cdn.elifesciences.org/articles/93517/elife-93517-fig5-data3-v2.zip
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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. …
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 …
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 …
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 |
