Palmitoylation Is Indispensable for Remorin to Restrict Tobacco Mosaic Virus Cell-to-Cell Movement in Nicotiana benthamiana
<p>Acyl-resin assisted capture (Acyl-RAC) to detect palmitoylation of NbREMs and their respective palmitoylation-defective mutants in <span class="html-italic">N. benthamiana</span> leaves. (<b>a</b>) NbREM1.1, (<b>b</b>) NbREM1.3, (<b>c</b>) NbREM1.5, (<b>d</b>) NbREM1.8, (<b>e</b>) NbREM1.1-C206A, (<b>f</b>) NbREM1.3-C177A, (<b>g</b>) NbREM1.5-C172A, (<b>h</b>) NbREM1.5-C172/175A, (<b>i</b>) NbREM1.8-C195A. (<b>j</b>) The relative palmitoylation levels of NbREMs and their respective palmitoylation-defective mutants. Data are mean ± SD (<span class="html-italic">n</span> = 3). Immunoblotting was performed using anti-flag antibody. NH<sub>2</sub>OH indicates presence (+) or absence (−) of hydroxylamine required for acyl group cleavage during the thiopropyl Sepharose 6b capture step. Thiopropyl Sepharose 6b enriched proteins were eluted and represent palmitoylated proteins (palmitoylation). Prior to thiopropyl Sepharose 6b capture the samples were removed as an input loading control (input).</p> "> Figure 2
<p>Protein accumulation assay comparison of NbREM1.1 and NbREM1.1-C206A (<b>a</b>), NbREM1.3 and NbREM1.3-C177A (<b>b</b>), NbREM1.8 and NbREM1.8-C195A (<b>c</b>), NbREM1.5 and NbREM1.5-C172A (<b>d</b>), NbREM1.5 and NbREM1.5-C172/175A (<b>e</b>) in <span class="html-italic">N. benthamiana</span> leaves. Data are mean ± SD (<span class="html-italic">n</span> = 4). Asterisks mark significant differences according to two-tailed Student’s <span class="html-italic">t</span>-test; * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; ns, no significant difference. The opposite halves of <span class="html-italic">N. benthamiana</span> leaves separately expressed flag-tagged NbREMs or their palmitoylation-defective mutants by agro-infiltration. Immunoblotting with anti-actin antibody was used as a loading control. Immunoblotting with anti-flag antibody was used to detect protein accumulation of NbREMs and its palmitoylation-defective mutants.</p> "> Figure 3
<p>Subcellular localization of GFP-NbREMs and their respective palmitoylation-defective mutants in RFP-H2B transgenic <span class="html-italic">N. benthamiana</span> protoplast cells. (<b>a</b>) GFP-NbREM1.1 and GFP-NbREM1.1-C206A, (<b>b</b>) GFP-NbREM1.3 and GFP-NbREM1.3-C177A, (<b>c</b>) GFP-NbREM1.8 and GFP-NbREM1.8-C195A, (<b>d</b>) GFP-NbREM1.5, GFP-NbREM1.5-C172A, and GFP-NbREM1.5-C172/175A. The Z-stacks of optical sections were constructed to view the localization in protoplast using ZEN Black software. GFP-NbREMs and their palmitoylation-defective mutants were transiently expressed in transgenic <span class="html-italic">N. benthamiana</span> expressing RFP-H2B, of which the nuclei was marked by red fluorescence. The released protoplasts were collected and examined by a Zeiss LSM 880 or 980 Airyscan<sup>TM</sup> upright laser scanning confocal microscope at 48 hpi. Bar, 20 μm.</p> "> Figure 4
<p>NbREM1.5 negatively regulates tobacco mosaic virus (TMV) cell-to-cell movement. (<b>a</b>) The effect of four NbREMs on TMV-GFP cell-to-cell movement at 4 days post-inoculation (dpi) and 8 dpi. TMV-GFP was co-agroinfiltrated with flag-NbREMs or scarlet-flag (a negative control) on the opposite halves of <span class="html-italic">N. benthamiana</span> leaves. The TMV-GFP infection foci fluorescence signals were observed at 4 dpi for upper row and 8 dpi for lower row. Leaves were captured under a portable UV lamp. Bar, 1 cm. (<b>b</b>) Statistical analyses of the effects of the four NbREMs on TMV-GFP cell-to-cell movement in the lower row of (<b>a</b>). Data are mean ± SEM (<span class="html-italic">n</span> = 50). Asterisks mark significant differences according to two-tailed Student’s <span class="html-italic">t</span>-test; ** <span class="html-italic">p</span> < 0.01; ns, no significant difference. (<b>c</b>) Detection of the infection foci of TMV-GFP in TRV-mCherry (control) and NbREM1.5 knock-down (TRV-NbREM1.5) <span class="html-italic">N. benthamiana</span> plant leaves. The TMV-GFP infection foci fluorescence signals were observed at 4 dpi under the portable UV lamp. (<b>d</b>) Statistical analyses of TMV-GFP infection foci area in TRV-mCherry (as a control) and NbREM1.5 knock-down (TRV-NbREM1.5) <span class="html-italic">N. benthamiana</span> plant leaves. Data are mean ± SEM (<span class="html-italic">n</span> = 133). Asterisks mark significant differences according to two-tailed Student’s <span class="html-italic">t</span>-test; *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 5
<p>Palmitoylation is indispensable for NbREM1.5 to negatively regulate TMV cell-to-cell movement. (<b>a</b>) NbREM1.5 interacts with TMV MP in vivo by BiFC. Reconstituted YFP signals were observed at 48 h post-inoculation (hpi) by fluorescence confocal microscope in <span class="html-italic">N. benthamiana</span> leaf epidermal cells. Bar, 20 μm. (<b>b</b>) Effect of NbREM1.5 and its palmitoylation-defective mutants NbREM1.5-C172/175A on cell-to-cell diffusion of GFP under co-expression with TMV-MP. GUS-flag was used as the negative control. Confocal images were taken at 48 hpi. Bar, 20 μm. (<b>c</b>) Statistical analyses of the effect of NbREM1.5 and NbREM1.5-C172/175A on cell-to-cell diffusion of GFP under co-expression with TMV-MP. Data are mean ± SD (<span class="html-italic">n</span> = 65). Asterisks mark significant differences and “ns” marks no significant difference according to two-tailed Student’s <span class="html-italic">t</span>-test; *** <span class="html-italic">p</span> < 0.001. (<b>d</b>) Effect of NbREM1.5-C172/175A on TMV-GFP cell-to-cell movement. TMV-GFP was co-agroinfiltrated with flag-NbREM1.5-C172/175A or scarlet-flag (as a negative control) on the opposite halves of <span class="html-italic">N. benthamiana</span> leaves. The TMV-GFP infection foci fluorescence signals were observed at 4 days post-inoculation under a portable UV lamp. Bar, 1 cm. (<b>e</b>) Statistical analyses of the effect of NbREM1.5-C172/175A on TMV-GFP cell-to-cell movement. Data are mean ± SEM (<span class="html-italic">n</span> = 45). Asterisks mark significant differences according to two-tailed Student’s <span class="html-italic">t</span>-test; ** <span class="html-italic">p</span> < 0.01.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Growth Conditions
2.2. RNA Extraction and Plasmid Construction
2.3. Agroinfiltration in N. benthamiana
2.4. Multiple Sequence Alignment and Phylogenetic Analysis
2.5. Reverse Transcription-Quantitative PCR (RT-qPCR)
2.6. Virus Inoculation and Detection
2.7. Acyl-Resin Assisted Capture (Acyl-RAC)
2.8. Immunoblotting and Antibodies
2.9. Bimolecular Fluorescence Complementation (BIFC) and Subcellular Localization Assays
2.10. Tobacco Rattle Virus (TRV)-Induced Gene Silencing
2.11. Callose Deposition Assay
2.12. Quantification and Statistical Analysis
3. Results
3.1. Group 1 Remorin Proteins Are Palmitoylated in N. benthamiana
3.2. Palmitoylation Contributes to Remorin Protein Accumulation
3.3. Palmitoylation Contributes to Plasma Membrane Localization for Remorin Proteins
3.4. NbREM1.5 Negatively Regulates TMV Cell-to-Cell Movement
3.5. NbREM1.5 Interacts with TMV-MP and Interferes with Its Ability for Expanding Plasmodesmata Size Exclusion Limit
3.6. Palmitoylation Is Required for NbREM1.5 to Inhibit TMV Cell-to-Cell Movement
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Ma, T.; Fu, S.; Wang, K.; Wang, Y.; Wu, J.; Zhou, X. Palmitoylation Is Indispensable for Remorin to Restrict Tobacco Mosaic Virus Cell-to-Cell Movement in Nicotiana benthamiana. Viruses 2022, 14, 1324. https://doi.org/10.3390/v14061324
Ma T, Fu S, Wang K, Wang Y, Wu J, Zhou X. Palmitoylation Is Indispensable for Remorin to Restrict Tobacco Mosaic Virus Cell-to-Cell Movement in Nicotiana benthamiana. Viruses. 2022; 14(6):1324. https://doi.org/10.3390/v14061324
Chicago/Turabian StyleMa, Tingting, Shuai Fu, Kun Wang, Yaqin Wang, Jianxiang Wu, and Xueping Zhou. 2022. "Palmitoylation Is Indispensable for Remorin to Restrict Tobacco Mosaic Virus Cell-to-Cell Movement in Nicotiana benthamiana" Viruses 14, no. 6: 1324. https://doi.org/10.3390/v14061324