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Single Amino Acid Substitutions in the Cucumber Mosaic Virus 1a Protein Induce Necrotic Cell Death in Virus-Inoculated Leaves without Affecting Virus Multiplication

Graduate School of Agricultural Science, Tohoku University, 468-1, Aramaki-Aza-Aoba, Sendai 980-0845, Japan
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Viruses 2020, 12(1), 91; https://doi.org/10.3390/v12010091
Received: 4 October 2019 / Revised: 8 January 2020 / Accepted: 9 January 2020 / Published: 13 January 2020
(This article belongs to the Section Viruses of Plants, Fungi and Protozoa)
When Arabidopsis thaliana ecotype Col-0 was inoculated with a series of reassortant viruses created by exchanging viral genomic RNAs between two strains of cucumber mosaic virus (CMV), CMV(Y), and CMV(H), cell death developed in the leaves inoculated with reassortant CMV carrying CMV(H) RNA1 encoding 1a protein, but not in noninoculated upper leaves. In general, cell death in virus-infected plants is a critical event for virus survival because virus multiplication is completely dependent on host cell metabolism. However, interestingly, this observed cell death did not affect either virus multiplication in the inoculated leaves or systemic spread to noninoculated upper leaves. Furthermore, the global gene expression pattern of the reassortant CMV-inoculated leaves undergoing cell death was clearly different from that in hypersensitive response (HR) cell death, which is coupled with resistance to CMV. These results indicated that the observed cell death does not appear to be HR cell death but rather necrotic cell death unrelated to CMV resistance. Interestingly, induction of this necrotic cell death depended on single amino acid substitutions in the N-terminal region surrounding the methyltransferase domain of the 1a protein. Thus, development of necrotic cell death might not be induced by non-specific damage as a result of virus multiplication, but by a virus protein-associated mechanism. The finding of CMV 1a protein-mediated induction of necrotic cell death in A. thaliana, which is not associated with virus resistance and HR cell death, has the potential to provide a new pathosystem to study the role of cell death in virus–host plant interactions. View Full-Text
Keywords: cell death; cucumber mosaic virus; hypersensitive response; methyltransferase domain; necrosis cell death; cucumber mosaic virus; hypersensitive response; methyltransferase domain; necrosis
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Tian, A.; Miyashita, S.; Ando, S.; Takahashi, H. Single Amino Acid Substitutions in the Cucumber Mosaic Virus 1a Protein Induce Necrotic Cell Death in Virus-Inoculated Leaves without Affecting Virus Multiplication. Viruses 2020, 12, 91.

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  • Supplementary File 1:

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  • Externally hosted supplementary file 1
    Link: https://firestorage.jp/download/3a9ee978071d8a1b92c7e0e3213e5b70d3714ab0
    Description: Table S1. Induction of cell death in CMV(HYY)-inoculated leaves of 94 ecotypes of Arabidopsis thaliana. Table S2. Primer list for in vitro transcribed cDNA to RNA1, RNA2 and RNA3 of CMV(H) in this study. Table S3. Primer list for construction of chimeric cDNA to CMV RNA1 in this study. Table S4. Primer list for nucleotide substitution in cDNA to CMV RNA1 in this study. Table S5. The 149 up-regulated genes in CMV(Y)-inoculated Col::RCY1 leaves showing HR cell death. Table S6. The 35 up-regulated genes common to CMV(HYY)-inoculated Col-0 leaves showing necrotic cell death and CMV(Y)-inoculated Col-0::RCY1 leaves showing HR cell death. Table S7. The 17 up-regulated genes in CMV(HYY)-inoculated Col-0 leaves showing necrotic cell death. Table S8. The 43 down-regulated genes in CMV(Y)-inoculated Col::RCY1 leaves showing HR cell death. Table S9. The 7 down-regulated genes common to CMV(HYY)-inoculated Col-0 leaves showing necrotic cell death and CMV(Y)-inoculated Col::RCY1 leaves showing HR cell death. Table S10. The 12 down-regulated genes in CMV(HYY)-inoculated Col-0 leaves showing necrotic cell death. Table S11. GO enrichment analysis of the 149 up-regulated genes in CMV(Y)-inoculated Col::RCY1 leaves showing HR cell death. Table S12. GO enrichment analysis of the 35 up-regulated genes common to CMV(HYY)-inoculated Col-0 leaves showing necrotic cell death and CMV(Y)-inoculated Col::RCY1 leaves showing HR cell death. Table S13. GO enrichment analysis of the 17 up-regulated genes in CMV(HYY)-inoculated Col-0 leaves showing necrotic cell death.
  • Externally hosted supplementary file 2
    Link: https://firestorage.jp/download/f51061335e648461b68a89e2b1d593bd69a618f5
    Description: Figure S1. Schematic diagrams of RNA genomes of the reassortant CMVs in the present study: CMV(HHY), CMV(HYY), CMV(YHH), CMV(YYH), CMV(YHY), and CMV(HYH). RNA1, RNA2, and RNA3 of CMV(Y) are shown in gray, and RNA1, RNA2, and RNA3 of CMV(Ho) are shown in black. Figure S2. Visualization of cell death in CMV(HYY)- or CMV(Y)-inoculated Arabidopsis thaliana Col-0 leaves. (A) HR cell death in CMV(Y)-inoculated A. thaliana Col::pRCY1-HA#12 at 3 dpi under bright field (upper panel) and trypan blue-staining (lower panel), were shown. (B) Cell death in three independent CMV(HYY)-inoculated Arabidopsis thaliana Col-0 leaves at 5 days after inoculation (dpi), HR cell death in three independent CMV(Y)-inoculated A. thaliana Col::pRCY1-HA#12 leaves at 3 dpi, and mock-inoculated A. thaliana Col leaves at 3 and 5 days, were visualized by trypan blue staining. Figure S3. Accumulation of CMV coat protein in CMV(HYY)- or CMV(Y)-inoculated Arabidopsis thaliana Col-0 leaves at 5 days after inoculation. Coat protein was quantitatively measured using ELISA. Vertical bars indicate the standard error of each mean for coat protein quantity in three independent samples. The same letter indicates that there is no significant difference at p <0.05 by LSD-test. Figure S4. Detection of CMV coat protein in extracts of virus-inoculated leaves and non-inoculated upper Arabidopsis thaliana Col-0 leaves. Extracts from fully expanded leaves of three independent Col-0 (number 1, 2, and 3) plants inoculated with reassortant CMV: CMV(H-I) and CMV(Y-I) were analyzed at 7 days after inoculation (dpi) using western blotting. Extracts from non-inoculated upper leaves of corresponding plants were also analyzed at 7 dpi using western blotting. RuBISCO protein is shown as an internal reference for protein quantity in each sample. CMV coat protein in CMV(Y)-inoculated leaves (P) and mock-inoculated leaves (N) was quantified as a positive and negative control, respectively. Figure S5. Detection of CMV coat protein in extracts of virus-inoculated leaves and non-inoculated upper Arabidopsis thaliana Col-0 leaves. Fully expanded leaves of three independent Col-0 plants (number 1, 2, and 3), which were inoculated with reassortant CMV: CMV(Y-GI) and CMV(H-GI), were analyzed at 7 days after inoculation (dpi) by western blotting. Extracts from non-inoculated upper leaves of the corresponding plants were also analyzed at 7 dpi by western blotting. RuBISCO protein is shown as an internal reference for protein quantity in each sample. CMV coat protein in CMV(Y)-inoculated leaves (P) and mock-inoculated leaves (N) was quantified as a positive and negative control, respectively. Figure S6. Detection of CMV coat protein in extracts of virus-inoculated Arabidopsis thaliana Col-0 leaves and non-inoculated upper leaves. (A) Fully expanded leaves of three independent Col-0 (number 1, 2, and 3) plants inoculated with reassortant CMVs CMV(Y-HV), CMV(H-HV), CMV(Y-TV), CMV(H-HL), CMV(Y-HL), and CMV(H-TV), respectively, were analyzed at 7 days after inoculation (dpi) by western blotting. (B) Non-inoculated upper leaves of the corresponding plants were analyzed at 7 dpi by western blotting. RuBISCO protein is shown as an internal reference for protein quantity in each sample. CMV coat protein in CMV(Y)-inoculated leaves (P) and mock-inoculated leaves (N) was quantified as a positive and negative control, respectively. Figure S7. Detection of CMV coat protein in extracts of virus-inoculated Arabidopsis thaliana Col-0 leaves and non-inoculated upper leaves. (A) Fully expanded leaves of three independent Col-0 (number 1, 2, and 3) plants inoculated with CMV(T29A), CMV(I49V), CMV(G54S), CMV(S298Q), CMV(F299R), and CMV(H310N), respectively, were analyzed at 7 days after inoculation (dpi) by western blotting. (B) Non-inoculated upper leaves of the corresponding plants were analyzed at 7 dpi by western blotting. RuBISCO protein is shown as an internal reference for protein quantity in each sample. CMV coat protein in CMV(Y)-inoculated leaves (P) and mock-inoculated leaves (N) was quantified as a positive and negative control, respectively. Figure S8. Gene ontology enrichment analysis for up-regulated 149 differentially expressed genes specific in CMV(Y)-inoculated Col::pRCY1-HA#12 leaves showing HR cell death. The top 10 GO enrichment terms in BP for the DEGs were shown. The number of genes in each category is equal to the dot size. The dot color represents the p.adjust value. Figure S9. Gene ontology enrichment analysis for up-regulated 35 genes in both CMV(HYY)-inoculated Col-0 leaves showing necrotic cell death and CMV(Y)-inoculated Col-0::pRCY1-HA#12 leaves showing HR cell death. The top 10 GO enrichment terms in BP for the DEGs were shown. The number of genes in each category is equal to the dot size. The dot color represents the p.adjust value. Figure S10. Gene ontology enrichment analysis for up-regulated 17 genes specific in CMV(HYY)-inoculated Col-0 leaves showing necrotic cell death. The GO enrichment terms in BP for the DEGs were shown. The number of genes in each category is equal to the dot size. The dot color represents the p.adjust value.
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