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A highly efficient grapevine mesophyll protoplast system for transient gene expression and the study of disease resistance proteins

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Abstract

Plant protoplasts constitute a versatile system for transient gene expression and have been widely used with several plant species for the functional characterization of genes and studies of diverse signaling pathways. However, such a system has not been developed for grapevine (Vitis vinifera L.) due to the challenges of large-scale isolation of viable grapevine protoplasts. Here, we report a simplified method for obtaining high yields and excellent viability of isolated protoplasts from young grapevine leaves. In addition, both the conditions for isolation and transfection of grapevine mesophyll protoplasts were modified, and the system was shown to be suitable for protein expression and studies of protein subcellular localization and protein–protein interactions. In addition, we heterologously and transiently expressed the Arabidopsis thaliana disease resistance protein RPW8.2, which has previously been reported to confer broad-spectrum resistance to several biotrophic pathogens in different plant families, as a fluorescent fusion protein in grapevine protoplasts. We observed that expression of the RPW8.2 fusion protein was induced in response to application of exogenous salicylic acid and following infection by the grapevine downy mildew pathogen, Plasmopara viticola. These results illustrate the potential of this highly efficient mesophyll protoplast system for transient gene expression and investigation of the activity of disease resistance proteins in grapevine.

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Abbreviations

BiFC:

Bimolecular fluorescence complementation

BSA:

Bovine serum albumin

CDS:

Coding sequence

EST:

Expressed sequence tag

FDA:

Fluorescein diacetate

GFP:

Green fluorescent protein

MES:

4-Morpholineethanesulfonic acid

MAP:

Mitogen-activated protein

MS:

Murashige and Skoog

PEG:

Polyethylene glycol

RPW8.2:

Resistance to powdery mildew 8.2

SA:

Salicylic acid

YFP:

Yellow fluorescent protein

hpt:

h post-treatment

hpi:

h post-inoculation

References

  • Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, Gomez-Gomez L, Boller T, Ausubel FM, Sheen J (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415(6875):977–983. doi:10.1038/415977a

    Article  CAS  PubMed  Google Scholar 

  • Bai Y, Han N, Wu JX, Yang YN, Wang JH, Zhu MY, Bian HW (2014) A transient gene expression system using barley protoplasts to evaluate microRNAs for post-transcriptional regulation of their target genes. Plant Cell Tissue Organ Cult 119:211–219. doi:10.1007/s11240-014-0527-z

    Article  CAS  Google Scholar 

  • Barbier M, Bessis R (1990) Isolation and culture of grapevine cv. Chardonnay leaf protoplasts. Euphytica 47:39–44

    Article  Google Scholar 

  • Bart R, Chern M, Park CJ, Bartley L, Ronald PC (2006) A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods. doi:10.1186/1746-4811-2-13

    PubMed  PubMed Central  Google Scholar 

  • Becker D, Brettschneider R, Lorz H (1994) Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. Plant J 5:299–307. doi:10.1046/j.1365-313X.1994.05020299.x

    Article  CAS  PubMed  Google Scholar 

  • Brezeanu A, Rosu A (1984) Isolation and culture of cell protoplasts from the mesophyll callus of Vitis vinifera L. Mansf Rev Roum Biol Biol Veget 29:33–37

    Google Scholar 

  • Chavez-Barcenas AT, Valdez-Alarcon JJ, Martinez-Trujillo M, Chen L, Xoconostle-Cazares B, Lucas WJ, Herrera-Estrella L (2000) Tissue-specific and developmental pattern of expression of the rice sps1 gene. Plant Physiol 124:641–653. doi:10.1104/pp.124.2.641

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen SB, Tao LZ, Zeng LR, Vega-Sanchez ME, Umemura K, Wang GL (2006) A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice. Mol Plant Pathol 7:417–427. doi:10.1111/j.1364-3703.2006.00346.x

    Article  CAS  PubMed  Google Scholar 

  • Chen J, Yi Q, Song Q, Gu Y, Zhang J, Hu Y, Liu H, Liu Y, Yu G, Huang Y (2015) A highly efficient maize nucellus protoplast system for transient gene expression and studying programmed cell death-related processes. Plant Cell Rep 34(7):1239–1251. doi:10.1007/s00299-015-1783-z

    Article  CAS  PubMed  Google Scholar 

  • Choi HI, Park HJ, Park JH, Kim S, Im MY, Seo HH, Kim YW, Hwang I, Kim SY (2005) Arabidopsis calcium-dependent protein kinase AtCPK32 interacts with ABF4, a transcriptional regulator of abscisic acid-responsive gene expression, and modulates its activity. Plant Physiol 139(4):1750–1761. doi:10.1104/pp.105.069757

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Craig W, Gargano D, Scotti N, Nguyen TT, Lao NT, Kavanagh TA, Dix PJ, Cardi T (2005) Direct gene transfer in potato: a comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts. Plant Cell Rep 24(10):603–611. doi:10.1007/s00299-005-0018-0

    Article  CAS  PubMed  Google Scholar 

  • Davey MR, Cocking E (1972) Uptake of bacteria by isolated higher plant protoplasts. Nature 239:455–456

    Article  Google Scholar 

  • Davey MR, Anthony P, Power JB, Lowe KC (2005) Plant protoplasts: status and biotechnological perspectives. Biotechnol Adv 23:131–171. doi:10.1016/j.biotechadv.2004.09.008

    Article  CAS  PubMed  Google Scholar 

  • De Sutter V, Vanderhaeghen R, Tilleman S, Lammertyn F, Vanhoutte I, Karimi M, Inze D, Goossens A, Hilson P (2005) Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells. Plant J 44(6):1065–1076. doi:10.1111/j.1365-313X.2005.02586.x

    Article  PubMed  Google Scholar 

  • Dekeyser RA, Claes B, Rycke RMUD, Habets ME, Montagu MCV, Caplan AB (1990) Transient gene expression in intact and organized rice tissues. Plant Cell 2(7):591–602

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dodds PN, Rathjen JP (2010) Plant immunity: towards an integrated view of plant-pathogen interactions. Nat Rev Genet 11:539–548. doi:10.1038/nrg2812

    Article  CAS  PubMed  Google Scholar 

  • Dong JJ, Kharb P, Teng WM, Hall TC (2001) Characterization of rice transformed via an Agrobacterium-mediated inflorescence approach. Mol Breed 7:187–194. doi:10.1023/A:1011357709073

    Article  CAS  Google Scholar 

  • Fontes N, Silva R, Vignault C, Lecourieux F, Geros H, Delrot S (2010) Purification and functional characterization of protoplasts and intact vacuoles from grape cells. BMC Res Notes 3:19. doi:10.1186/1756-0500-3-19

    Article  PubMed  PubMed Central  Google Scholar 

  • Guo JJ, Morrell-Falvey JL, Labbe JL, Muchero W, Kalluri UC, Tuskan GA, Chen JG (2012) Highly efficient isolation of populus mesophyll protoplasts and its application in transient expression assays. PLoS One. doi:10.1371/journal.pone.0044908

    Google Scholar 

  • Hoffman A, Halfter U, Morris PC (1994) Transient expression in leaf mesophyll protoplasts of Arabidopsis-thaliana. Plant Cell Tissue Organ Cult 36:53–58. doi:10.1007/Bf00048315

    Article  CAS  Google Scholar 

  • Hong SY, Seo PJ, Cho SH, Park CM (2012) Preparation of leaf mesophyll protoplasts for transient gene expression in Brachypodium distachyon. J Plant Biol 55:390–397. doi:10.1007/s12374-012-0159-y

    Article  CAS  Google Scholar 

  • Kovtun Y, Chiu WL, Zeng WK, Sheen J (1998) Suppression of auxin signal transduction by a MAPK cascade in higher plants. Nature 395:716–720

    Article  CAS  PubMed  Google Scholar 

  • Kovtun Y, Chiu WL, Tena G, Sheen J (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc Natl Acad Sci USA 97:2940–2945. doi:10.1073/pnas.97.6.2940

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kurth EG, Peremyslov VV, Prokhnevsky AI, Kasschau KD, Miller M, Carrington JC, Dolja VV (2012) Virus-derived gene expression and RNA interference vector for grapevine. J Virol 86:6002–6009. doi:10.1128/Jvi.00436-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee SM, Hoang MHT, Han HJ, Kim HS, Lee K, Kim KE, Kim DH, Lee SY, Chung WS (2009) Pathogen inducible voltage-dependent anion channel (AtVDAC) isoforms are localized to mitochondria membrane in Arabidopsis. Mol Cells 27(3):321–327. doi:10.1007/s10059-009-0041-z

    Article  CAS  PubMed  Google Scholar 

  • Li JF, Park E, von Arnim AG, Nebenfuhr A (2009) The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods. doi:10.1186/1746-4811-5-6

    PubMed  PubMed Central  Google Scholar 

  • Liu CN, Li XQ, Gelvin SB (1992) Multiple copies of virg enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens. Plant Mol Biol 20:1071–1087. doi:10.1007/Bf00028894

    Article  CAS  PubMed  Google Scholar 

  • Manavella PA, Chan RL (2009) Transient transformation of sunflower leaf discs via an Agrobacterium-mediated method: applications for gene expression and silencing studies. Nat Protoc 4:1699–1707. doi:10.1038/nprot.2009.178

    Article  CAS  PubMed  Google Scholar 

  • Marion J, Bach L, Bellec Y, Meyer C, Gissot L, Faure JD (2008) Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings. Plant J 56:169–179. doi:10.1111/j.1365-313X.2008.03596.x

    Article  CAS  PubMed  Google Scholar 

  • Mii M, Zou YM, Sugiyama T, Yanagihara S, Iizuka M (1991) High-frequency callus formation from protoplasts of Vitis labruscana Bailey and Vitis thunbergii Sieb. et Zucc. by embedding in gellan gum. Sci Hortic. 46:253–260

    Article  Google Scholar 

  • Myles S, Boyko AR, Owens CL, Brown PJ, Grassi F, Aradhya MK, Prins B, Reynolds A, Chia JM, Ware D, Bustamante CD, Buckler ES (2011) Genetic structure and domestication history of the grape. Proc Natl Acad Sci USA 108(9):3530–3535. doi:10.1073/pnas.1009363108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nicolia A, Proux-Wera E, Ahman I, Onkokesung N, Andersson M, Andreasson E, Zhu LH (2015) Targeted gene mutation in tetraploid potato through transient TALEN expression in protoplasts. J Biotechnol 204:17–24. doi:10.1016/j.jbiotec.2015.03.021

    Article  CAS  PubMed  Google Scholar 

  • Nunan KJ, Sims IM, Bacic A, Robinson SP, Fincher GB (1997) Isolation and characterization of cell walls from the mesocarp of mature grape berries (Vitis vinifera). Planta 203(1):93–100. doi:10.1007/s004250050169

    CAS  Google Scholar 

  • Ohyama K, Gamborg OL, Miller RA (1972) Uptake of exogenous DNA by plant protoplasts. Can J Bot 50:2077–2080

    Article  CAS  Google Scholar 

  • Reustle G, Natter I (1994) Effect of polyvinylpyrrolidone and activated-charcoal on formation of microcallus from grapevine protoplasts (Vitis sp). Vitis 33:117–121

    CAS  Google Scholar 

  • Reustle G, Harst M, Alleweldt G (1995) Plant regeneration of grapevine (Vitis sp) protoplasts isolated from embryogenic tissue. Plant Cell Rep 15:238–241

    CAS  PubMed  Google Scholar 

  • Sheen J (2001) Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol 127:1466–1475. doi:10.1104/Pp.010820

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sheen J, Hwang SB, Niwa Y, Kobayashi H, Galbraith DW (1995) Green-fluorescent protein as a new vital marker in plant-cells. Plant J 8:777–784. doi:10.1046/j.1365-313X.1995.08050777.x

    Article  CAS  PubMed  Google Scholar 

  • Skene KG (1974) Culture of protoplasts from grapevine pericarp callus. Aust. J. Plant. Physiol. 1:371–376

    Article  Google Scholar 

  • Skene KG (1975) Production of callus from protoplasts of cultured grape pericarp. Vitis 14:177–180

    Google Scholar 

  • Swanson SJ, Bethke PC, Jones RL (1998) Barley aleurone cells contain two types of vacuoles: characterization of lytic organelles by use of fluorescent probes. Plant Cell 10:685–698

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tan BY, Xu M, Chen Y, Huang MR (2013) Transient expression for functional gene analysis using Populus protoplasts. Plant Cell Tissue Organ Cult 114:11–18. doi:10.1007/s11240-013-0299-x

    Article  CAS  Google Scholar 

  • Theodoropoulos PA, Roubelakis-Angelakis KA (1990) Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L. Plant Cell Tissue Organ Cult 20:15–23

    Article  Google Scholar 

  • Ueki S, Lacroix B, Krichevsky A, Lazarowitz SG, Citovsky V (2009) Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment. Nat Protoc 4:71–77. doi:10.1038/nprot.2008.217

    Article  CAS  PubMed  Google Scholar 

  • Waadt R, Schmidt LK, Lohse M, Hashimoto K, Bock R, Kudla J (2008) Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. Plant J 56:505–516. doi:10.1111/j.1365-313X.2008.03612.x

    Article  CAS  PubMed  Google Scholar 

  • Wang WM, Devoto A, Turner JG, Xiao SY (2007) Expression of the membrane-associated resistance protein RPW8 enhances basal defense against biotrophic pathogens. Mol Plant Microbe Interact 20:966–976. doi:10.1094/Mpmi-20-8-0966

    Article  CAS  PubMed  Google Scholar 

  • Wang WM, Wen YQ, Berkey R, Xiao SY (2009) Specific targeting of the Arabidopsis resistance protein RPW8.2 to the interfacial membrane encasing the fungal haustorium renders broad-spectrum resistance to powdery mildew. Plant Cell 21:2898–2913. doi:10.1105/tpc.109.067587

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang HL, Wang W, Zhan JC, Huang WD, Xu HY (2015) An efficient PEG-mediated transient gene expression system in grape protoplasts and its application in subcellular localization studies of flavonoids biosynthesis enzymes. Sci Hortic 191:82–89. doi:10.1016/j.scienta.2015.04.039

    Article  CAS  Google Scholar 

  • Widholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol 47:189–194

    Article  CAS  PubMed  Google Scholar 

  • Xiao SY, Ellwood S, Calis O, Patrick E, Li TX, Coleman M, Turner JG (2001) Broad-spectrum mildew resistance in Arabidopsis thaliana mediated by RPW8. Science 291:118–120. doi:10.1126/science.291.5501.118

    Article  CAS  PubMed  Google Scholar 

  • Xiao SY, Brown S, Patrick E, Brearley C, Turner JG (2003a) Enhanced transcription of the Arabidopsis disease resistance genes RPW8.1 and RPW8.2 via a salicylic acid-dependent amplification circuit is required for hypersensitive cell death. Plant Cell 15:33–45. doi:10.1105/tpc.006940

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xiao SY, Charoenwattana P, Holcombe L, Turner JG (2003b) The Arabidopsis genes RPW8.1 and RPW8.2 confer induced resistance to powdery mildew diseases in tobacco. Mol Plant Microbe Interact 16:289–294. doi:10.1094/Mpmi.2003.16.4.289

    Article  CAS  PubMed  Google Scholar 

  • Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572. doi:10.1038/nprot.2007.199

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Su J, Duan S, Ao Y, Dai J, Liu J, Wang P, Li Y, Liu B, Feng D, Wang J, Wang H (2011) A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 7(1):30. doi:10.1186/1746-4811-7-30

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang HF, Liu WZ, Zhang YP, Deng M, Niu FF, Yang B, Wang XL, Wang BY, Liang WW, Deyholos MK, Jiang YQ (2014a) Identification, expression and interaction analyses of calcium-dependent protein kinase (CPK) genes in canola (Brassica napus L.). BMC Genomics. doi:10.1186/1471-2164-15-211

    Google Scholar 

  • Zhang HF, Yang B, Liu WZ, Li HW, Wang L, Wang BY, Deng M, Liang WW, Deyholos MK, Jiang YQ (2014b) Identification and characterization of CBL and CIPK gene families in canola (Brassica napus L.). BMC Plant Biol. doi:10.1186/1471-2229-14-8

    Google Scholar 

  • Zhang K, Han YT, Zhao FL, Hu Y, Gao YR, Ma YF, Zheng Y, Wang YJ, Wen YQ (2015) Genome-wide Identification and Expression Analysis of the CDPK Gene Family in Grape, Vitis spp. Bmc Plant Biol. doi:10.1186/S12870-015-0552-Z

    Google Scholar 

  • Zhou XN, Yuan FF, Wang MY, Guo AG, Zhang YF, Xie CG (2013) Molecular characterization of an ABA insensitive 5 orthologue in Brassica oleracea. Biochem Biophys Res Commun 430:1140–1146. doi:10.1016/j.bbrc.2012.12.023

    Article  CAS  PubMed  Google Scholar 

  • Zhu Y, Guo D, Li G (1993) Studies of several factors in grape protoplast culture. J Northeast Agric Coll 24:17–19

    Google Scholar 

  • Zhu YM, Hoshino Y, Nakano M, Takahashi E, Mii M (1997) Highly efficient system of plant regeneration from protoplasts of grapevine (Vitis vinifera L.) through somatic embryogenesis by using embryogenic callus culture and activated charcoal. Plant Sci 123:151–157. doi:10.1016/S0168-9452(96)04557-8

    Article  CAS  Google Scholar 

  • Zubko MK, Zubko EI, van Zuilen K, Meyer P, Day A (2004) Stable transformation of petunia plastids. Transgenic Res 13:523–530. doi:10.1007/s11248-004-2374-x

    Article  CAS  Google Scholar 

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Acknowledgments

We thank YQJ, CGX and SX kindly provided the vectors of BiFC, BolABI5 and RPW8.2, respectively. The authors would also like to thank two anonymous reviewers for comments on the manuscript. This work was supported by the National Natural Science Foundation of China (Grant Nos. 31372022, 31071772), the Shaanxi province science and technology research and development Program (2014K02-02-03) and the Fundamental Research Funds for the Central Universities (2452015140).

Author contributions

Y.Q.W. conceived the research. F.L.Z. performed all treatments with assistance of Y.H., Y.J.L., Y.R.G., X.W.Z., and Q.D. Y.H. and Y.R.G. carried out partly subcellular localization experiments. Y.J.L. prepared all plant materials. Y.Q.W., F.L.Z. and Y.H. analysed and interpreted the data. Y.J.W. contributed with consultation. F.L.Z. wrote the manuscript and Y.Q.W. revised it. All authors read and approved the final manuscript.

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Authors and Affiliations

  1. State Key Laboratory of Crop Stress Biology for Arid Areas and College of Horticulture, Northwest A&F University, Yangling, 712100, Shaanxi, China

    Feng-Li Zhao, Ya-Juan Li, Yang Hu, Yu-Rong Gao, Xue-Wen Zang, Qin Ding, Yue-Jin Wang & Ying-Qiang Wen

  2. Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest China, Ministry of Agriculture, Yangling, 712100, Shaanxi, China

    Feng-Li Zhao, Ya-Juan Li, Yang Hu, Yu-Rong Gao, Xue-Wen Zang, Qin Ding, Yue-Jin Wang & Ying-Qiang Wen

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  1. Feng-Li Zhao
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Correspondence to Ying-Qiang Wen.

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Table S1

Primers used in this study. (DOC 34 kb)

Fig. S1

Isolation of grapevine mesophyll protoplasts. a The effect of the amount of leaf tissue used on protoplast production imaged by bright field and using fluorescence microscopy when fluorescein diacetate (FDA) stained for viability (green). b The effect of digestion time and concentration of mannitol on protoplast viability as visualized by FDA staining (green). Scale bars = 50 μm. (TIFF 1794 kb)

Fig. S2

Isolation of protoplasts from grapevine leaves using different enzyme cocktails. Fluorescein diacetate (FDA) staining was used to determine the viability of protoplasts. C, cellulase R-10; M, macerozyme R-10. Scale bars = 50 μm. (TIFF 797 kb)

Fig. S3

A healthy two-month old V. vinifera cv. Rizamat plant grown in a controlled environment chamber for use in protoplast isolation. a The numbers represent the position of the leaves. b Effect of leaf position from tissue cultured plants, plants grown in the controlled chambers and plants from the greenhouse on protoplast yield. Scale bars represent 3 cm. (TIFF 967 kb)

Fig. S4

Transient gene expression in grapevine protoplasts. Effect of concentration of protoplasts (a) and incubation time (b) with polyethylene glycol (PEG) on transfection efficiency. Fluorescent (left panels) and bright field (right panels) microscopic images were taken. Scale bars = 50 μm. (TIFF 2778 kb)

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Zhao, FL., Li, YJ., Hu, Y. et al. A highly efficient grapevine mesophyll protoplast system for transient gene expression and the study of disease resistance proteins. Plant Cell Tiss Organ Cult 125, 43–57 (2016). https://doi.org/10.1007/s11240-015-0928-7

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