Abstract
Grapevine (Vitis vinifera L.) is an economically important fruit crop cultivated worldwide. In China, grapevine cultivation is very extensive, and a few Vitis grapes have excellent pathogen and stress resistance, but the molecular mechanisms underlying the grapevine response to stress remain unclear. In this study, a microRNA (miRNA; miR827a), which negatively regulates its target gene VqMYB14, a key regulatory role in the synthesis of stilbenes, was identified in Vitis quinquangularis (V. quinquangularis) using transcriptome sequencing. Using overexpression and silencing approaches, we found that miR827a regulates the synthesis of stilbenes by targeting VqMYB14. We used flagellin N-terminal 22-amino-acid peptide (flg22), the representative elicitor in plant basal immunity, as the elicitor to verify whether miR827a is involved in the basal immunity of V. quinquangularis. Furthermore, the promoter activity of miR827a was alleviated in transgenic grape protoplasts and Arabidopsis thaliana following treatment with flg22 and Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000), respectively. In addition, yeast one-hybrid and dual luciferase reporter assay revealed that the ethylene transcription factor VqERF057 acted as a key regulator in the inhibition of miR827a transcription. These results will contribute to the understanding of the biological functions of miR827a in grapevine and clarify the molecular mechanism of the interaction between miR827a and VqMYB14.
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Acknowledgements
This research was supported by the National Natural Science Foundation of China (31970348 and 31600256), the International Scientific and Technological Cooperation Projects of Shaanxi Province (2022KW-45), and the Young Academic Talent Support Program of Northwest University and Graduate and Innovation Program of Northwest University (CX2023053).
Funding
This work was supported by the National Natural Science Foundation of China (31970348 and 31600256), the International Scientific and Technological Cooperation Projects of Shaanxi Province (2022KW-45), and the Young Academic Talent Support Program of Northwest University and GraduateInnovation Program of Northwest University (CX2023053).
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DD conceived and designed the work. YY-L performed the major parts of the experiments. LX-W, JZ, JW-T, LY, QL, JL, and QY performed some of the experiments. YY-L analysed the data and wrote the manuscript. DD revised the manuscript. All authors gave final approval for the submission of the paper.
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122_2024_4599_MOESM1_ESM.pptx
Fig. S1. (A) RT-qPCR analysis of miR827a and VqMYB14 expression in leaves of V. quinquangularis were treated with Al3+ and flg22 for 12 h. 5.8s rRNA (for miR827a) and VvGAPDH (for VqMYB14) were used as the internal control. The difference between the Ct values of the target gene X and those for the reference R was calculated as follows: ΔCt (X) =Ct (X)–Ct (R). The final result was expressed as 2–ΔΔCt(X). Data are means ± SE of three biological replicates. Statistical significance was calculated using Student’s t test (*P <0.05 and **P <0.01). (B) Secondary structure of the miR827a precursor and the location of mature miR827a (purple). (C) The picture of Cabernet Sauvignon protoplasts under a 20x microscope. (PPTX 401 KB)
122_2024_4599_MOESM2_ESM.pptx
Fig. S2. Overexpression and interference of VqMYB14 and its effects on VqSTS48 and stilbene accumulation in grapevine leaves. (A) RT-qPCR analysis of VqSTS48 expression in leaves of V. quinquangularis were infiltrated with Agrobacterium harbouring the pCAMBIA1301 empty vector (Control 1) or overexpressed construct OE-VqMYB14 and pBI121 empty vector (Control 2) or VqMYB14RNAi. (B) Stilbenes were extracted from OE-VqMYB14 or control 1 and VqMYB14RNAi or control 2 leaves and then quantified by high-performance liquid chromatography (HPLC) analysis (DaoJin LC-20A, Japan). (C) RT-qPCR analysis of miR827a and VqMYB14 expression in leaves of STTM-miR827a or pBWA(V)KS empty vector (control 3) and VqMYB14RNAi or control 2. EF1-α (for mRNAs) and miR168 (for miR827a) were used as the internal control. The difference between the Ct values of the target gene X and those for the reference R was calculated as follows: ΔCt (X) =Ct (X)–Ct (R). The final result was expressed as 2–ΔΔCt(X).Data are means ± SE of three biological replicates. Statistical significance was calculated using Student’s t test (*P <0.05 and **P <0.01). (PPTX 184 KB)
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Fig. S3. Comparison sequences of mature sequence miR827a (inclusing its upstream 172 bp and downstream 107 bp) (A) and corresponding target site MYB14 (including its upstream 171 bp and downstream 196 bp) (B) in V. vinifera cv. Cabernet Sauvignon, Carignan, V. labrusca cv.Concord, V. rotundifolia Michx, V. davidii, and V. quinquangularis. (PPTX 750 KB)
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Fig. S4. RT-qPCR analysis of VqSTS48 expressions in transgenic Arabidopsis treated by flg22 60 h. Actin8 (A) and Actin2 (B) were used as the internal controls, respectively. Data are means ± SE of three biological replicates. The difference between the Ct values of the target gene X and those for the reference R was calculated as follows: ΔCt (X) =Ct (X)–Ct (R). The final result was expressed as 2–ΔΔCt(X). Different letters indicate significant differences by one-way ANOVA according to Duncan’s test (P <0.05). (PPTX 239 KB)
122_2024_4599_MOESM5_ESM.pptx
Fig. S5. Relative Fluc/Rluc activity in Cabernet Sauvignon protoplasts co-transfected with miR827a and VqMYB14-CDSwt (Control: VqMYB14-CDSwt + pBWA(V)HS empty vector; miR827a: VqMYB14-CDSwt + OE-miR827a) or VqMYB14-CDS mut (mutant) (Control: VqMYB14-CDSmut + pBWA(V)HS empty vector; miR827a: VqMYB14-CDSmut + OE-miR827a). Data are means ± SE of three biological replicates. Statistical significance was calculated using Student’s t test (*P <0.05). (PPTX 94 KB)
122_2024_4599_MOESM15_ESM.docx
Table S10. RT-qPCR analysis of miR827a and VqMYB14 expressions by transient overexpression of miR827a in V. quinquangularis. (DOCX 16 KB)
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Luo, Y., Wang, L., Zhu, J. et al. The grapevine miR827a regulates the synthesis of stilbenes by targeting VqMYB14 and gives rise to susceptibility in plant immunity. Theor Appl Genet 137, 95 (2024). https://doi.org/10.1007/s00122-024-04599-9
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DOI: https://doi.org/10.1007/s00122-024-04599-9