Abstract
An efficient agroinfiltration-based gene silencing assay was established to evaluate candidate genes likely to be involved in resistance to powdery mildew (Uncinula necator) in grapevine (Vitis vinifera L.). Functional assays were performed using two grapevine genotypes, cv. Superior, mildew-susceptible, and cv. Run1 Mtp3294, mildew-resistant. Constructs encoding a self-complementary “hairpin” RNA for phytoene desaturase (PDS) along with the green fluorescent protein gene were introduced into Agrobacterium tumefaciens. These constructs were used for agroinfiltration of leaf tissues, yielding both efficient transformation and gene silencing events. The agroinfiltration procedure did not interfere with the powdery mildew infection steps. Confocal laser-scanning microscopy analysis revealed co-localization of the GFP signal with cells lacking chlorophyll autofluorescence, further supporting PDS gene silencing. The extent of PDS mRNA degradation was evaluated by qRT-PCR in the agroinfiltrated areas and results supported a significant reduction of the gene transcripts in the silenced regions. Findings of the present work suggest that the developed silencing procedures represent a useful tool for functional characterization of grapevine genes involved in powdery mildew resistance.
Similar content being viewed by others
References
Becker A, Lange M (2009) VIGS-genomics goes functional. Trends Plant Sci 15:1–4
Bhaskar PB, Venkateshwaran V, Wu L, Anè J-M, Jiang J (2009) Agrobacterium-mediated transient gene expression and silencing: a rapid tool for functional gene assay in potato. PLoS One 4(6):e5812. doi:10.1371/journal.pone.0005812
Bolte S, Talbot C, Boutte Y, Catrice O, Read ND, Satiat-Jeunemaitre B (2004) FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells. J Microsc 214:159–173
Bouquet A (1986) Introduction dans l’espece Vitis vinifera L. d’un caractere de resistance a l’oidium (Uncinula necator Schw. Burr.) issu de l’espece Muscadinia rotundifolia (Michx.) Small. Vigne-vini 12:141–146
Brodersen P, Voinnet O (2006) The diversity of RNA silencing pathways in plants. Trends Genet 22(5):268–280
Carra A, Gambino G, Urso S, Nervo G (2011) Non coding RNAs and gene silencing in grape. In: Erdmann VA, Barciszewski J (eds) Non coding RNAs in plants, RNA technologies. Springer, Berlin, pp 67–78
Cunningham FX Jr, Gantt E (1998) Genes and enzymes of carotenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 49:557–583
Dong W, Nowara D, Schweizer P (2006) Protein polyubiquitination plays a role in basal host resistance of barley. Plant Cell 18:3321–3331
Douchkov D, Nowara D, Zierold U, Schweizer P (2005) A high-throughput gene-silencing system for the functional assessment of defence-related genes in barley epidermal cells. MPMI 18:755–761
Dry IB, Feechan A, Anderson C, Jermakow AM, Bouquet A, Adam-Blondon A-F, Thomas MR (2010) Molecular strategies to enhance the genetic resistance of grapevines to powdery mildew. Austr J Grape Wine Res 16:94–105
Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song K, Pikaard CS (2006) Gateway compatible vectors for plant functional genomics and proteomics. Plant J 45:616–629
Faccioli P, Stanca AM, Morcia C, Alberici R, Terzi V (2010) Identification of a set of widely expressed genes in grape (Vitis vinifera L.) and its functional characterization: a multi-evidence based study. Vitis 49(4):175–179
Frizzi A, Huang S (2010) Tapping RNA silencing pathways for plant biotechnology. Plant Biotechnol J 8:1–23
Hein I, Barciszewska-Pacak M, Hrubikova K, Williamson S, Dinesen M, Soenderby IE, Sundar S, Jarmolowski A, Shirasu K, Lacomme C (2005) Virus-induced gene silencing-based functional characterization of genes associated with powdery mildew resistance in barley. Plant Physiol 138:2155–2164
Igarashi A, Yamagata K, Sugai T, Takahashi Y, Sugawara E, Tamura A, Yaegashi H, Yamagishi N, Takahashi T, Isogai M, Takahashi H, Yoshikawa N (2009) Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes. Virology 386:407–416
Jaillon O, Aury J-M, Noel B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, Vitulo N et al (2007) The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449:463–467. doi:10.1038/nature06148
Kankanala P, Czymmek K, Valenta B (2007) Roles for rice membrane dynamics and plasmodesmata during biotrophic invasion by the blast fungus. Plant Cell 19:706–724
Karimi M, Inzè D, Depicker A (2002) GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195
Karimi M, Depicker A, Hilson P (2007) Recombinational cloning with plant Gateway vectors. Plant Physiol 145:1144–1154
Leinhos GME, Gold RE, Düggelin M, Guggenheim R (1997) Development and morphology of Uncinula necator following treatment with the fungicides kresoxim-methyl and penconazole. Mycol Res 101:1033–1046
Mani T, Manjula S (2011) Optimization of Agrobacterium-mediated transient gene expression and endogenous gene silencing in Piper colubrinum Link. by vacuum infiltration. Plant Cell Tiss Organ Cult 105:113–119
Martin RC, Glover-Cutter K, Martin RR, Dombrowski JE (2012) Virus induced gene silencing in Lolium temulentum. Plant Cell Tiss Organ Cult. doi:10.1007/s11240-012-0257-z
McGinnis KM (2010) RNAi for functional genomics in plants. Brief Funct Genomics 9:111–117
Muruganantham M, Moskovitz Y, Haviva S, Horesha T, Fenigsteina A, Preezb J, Stephanb D, Burger JT, Mawassi M (2009) Grapevine virus A-mediated gene silencing in Nicotiana benthamiana and Vitis vinifera. J Virol Methods 155:167–174
Niks RE (1986) Failure of haustorial development as a factor in slow growth and development of Puccinia hordei in partially resistant barley seedlings. Physiol Mol Plant Pathol 28:309–322
Pauquet J, Bouquet A, This P, Adam-Blondon A-F (2001) Establishment of a local map of AFLP markers around the powdery mildew resistance gene Run1 in grapevine and assessment of their usefulness for marker assisted selection. Theor Appl 103:1201–1210
Payne RW, Harding SA, Murray DA, Soutar DM, Baird DB, Welham SJ, Kane AF, Gilmour AR, Thompson R, Webster R, Tunnicliffe Wilson G (2006) GenStat release 9 reference manual, part 2 directives. VSN International, Hemel Hempstead
Pearson RC, Gadoury DM (1991) Powdery mildew of grape. In: Kumar J, Chaube HS, Singh US, Mukhopadhyay AN (eds) Plant diseases of international importance: diseases of fruit crops, vol III. Prentice Hall, Englewood Cliffs, pp 129–146
Pruss GJ, Nester EW, Vance V (2008) Infiltration with Agrobacterium tumefaciens induces host defense and development-dependent responses in the infiltrated zone. MPMI 21:1528–1538
Romero I, Tikunov Y, Bovy A (2011) Virus-induced gene silencing in detached tomatoes and biochemical effects of phytoene desaturase gene silencing. J Plant Physiol 168:1129–1135
Rumbolz J, Kassermeyer H-H, Steinmetz V, Deising HB, Mendgen K, Mathys D, Wirtz S, Guggenheim R (2000) Differentiation of infection structures of the powdery mildew fungus Uncinula necator and adhesion to the host cuticle. Can J Bot 78:409–421
Santos-Rosa M, Poutaraud A, Merdinoglu D, Mestre P (2008) Development of a transient expression system in grapevine via agro-infiltration. Plant Cell Rep 27:1053–1063
Tanaka S, Ishihama N, Yoshioka H, Huser A, O’Connell R, Tsuji G, Tsuge S, Kubo Y (2009) The Colletotrichum orbiculare ssd1 mutant enhances Nicotiana benthamiana basal resistance by activating a mitogen-activated protein kinase pathway. Plant Cell 21:2517–2526
Varsha Wesley S, Helliwell CA, Smith NA, Wang M, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001) Construct design for efficient, effective and highthroughput gene silencing in plants. Plant J 27:581–590
Velasco R, Zharkikh A, Troggio M, Cartwright DA, Cestaro A et al (2007) A high quality draft consensus sequence of the genome of a heterozygous grapevine variety. PLoS One 2(12):e1326. doi:10.1371/journal.pone.0001326
Vidal JR, Gomez C, Cutanda MC, Shrestha BR, Bouquet A, Thomas MR, Torregrosa L (2010) Use of gene transfer technology for functional studies in grapevine. Aust J Grape Wine Res 16:138–151
Wielopolska A, Townley H, Moore I, Waterhouse P, Helliwell C (2005) A high-throughput inducible RNAi vector for plants. Plant Biotechnol J 3:583–590
Wroblewski T, Tomczak A, Michelmore R (2005) Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnol J 3:259–273
Zottini M, Barizza E, Costa A, Formentin E, Ruberti C, Carimi F, Lo Schiavo F (2008) Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells. Plant Cell Rep 27:845–853
Acknowledgments
This work was supported by the Italian national project ‘VItis GeNome Analysis’ (‘VIGNA’), Progetto di Ateneo and Progetto from Agriculture Ministry and funded by MiPAF (Ministero delle Politiche Agricole e Forestali). We thank Dr. Anne-Françoise Adam-Blondon and Mrs. Marion Guillou, INRA (France) for providing the Run1 Mtp3294 grapevine genotype and Dr Mark R. Thomas supplying the PDS EST clone (GeneBank ID: CN007512). We thank Donata Pagani (CRA-GPG) for excellent technical assistance, Dr Alessandro Tondelli for statistical analysis and Dr Elisabetta Barizza for hydroponic plants management.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Urso, S., Zottini, M., Ruberti, C. et al. An Agrobacterium tumefaciens-mediated gene silencing system for functional analysis in grapevine. Plant Cell Tiss Organ Cult 114, 49–60 (2013). https://doi.org/10.1007/s11240-013-0305-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-013-0305-3