Abstract
One major threat to plant cultivation are fungal pathogens, which can cause substantial yield losses in agriculture. As an example, cereal powdery mildew fungi such as the barley (Hordeum vulgare) pathogen, Blumeria graminis f. sp. hordei (Bgh), are among the ten most relevant fungal plant pathogens in molecular plant pathology and can lead to yield losses of up to 30%. Plant Mildew resistance Locus O (MLO) genes are required for successful colonization of plants by powdery mildew fungi. Accordingly, loss-of-function mlo mutants confer durable resistance against powdery mildew fungi in many plant species. In the case of barley, mlo-based resistance has been used for more than 40 years in agriculture without powdery mildew fungi effectively overcoming this kind of immunity. However, the molecular basis of mlo resistance and function(s) of the transmembrane Mlo protein(s) are still incompletely understood. The generation of transgenic barley plants to study the plant immune response and the involvement of Mlo therein is time-consuming and challenging. Therefore, transient gene expression via gene gun-mediated particle bombardment became a popular, easy, and efficient tool to investigate different aspects of plant defense responses in barley. Since Bgh fails to penetrate leaf epidermal cells of mlo mutants, single-cell complementation upon biolistic transformation resulting in (over-)expression of Mlo can be used to characterize the Mlo protein functionally in vivo. In this chapter, we describe in detail the gene gun-mediated transient expression of Mlo in barley leaf epidermal cells followed by powdery mildew inoculation and the subsequent microscopic evaluation. However, gene gun-mediated transient gene expression may be also used to address other research questions or to transform the epidermal tissues of other plant organs and/or species.
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References
Kusch S, Pesch L, Panstruga R (2016) Comprehensive phylogenetic analysis sheds light on the diversity and origin of the MLO family of integral membrane proteins. Genome Biol Evol 8(3):878–895
Acevedo-Garcia J, Kusch S, Panstruga R (2014) Magical mystery tour: MLO proteins in plant immunity and beyond. New Phytol 204(2):273–281
Büschges R et al (1997) The barley Mlo gene: a novel control element of plant pathogen resistance. Cell 88(5):695–705
Jørgensen IH (1992) Discovery, characterization and exploitation of Mlo powdery mildew resistance in barley. Euphytica 63:141–152
Consonni C et al (2006) Conserved requirement for a plant host cell protein in powdery mildew pathogenesis. Nat Genet 38(6):716–720
Kusch S, Panstruga R (2017) mlo-based resistance: an apparently universal “weapon” to defeat powdery mildew disease. Mol Plant-Microbe Interact 30(3):1–47
Lyngkjær MF, Newton AC, Atzema JL, Baker SJ (2000) The barley mlo-gene: an important powdery mildew resistance source. Agronomie 20(7):745–755
Devoto A, Piffanelli P, Nilsson I, Wallin E, Panstruga R, Von Heijne G, Schulze-Lefert P (1999) Topology, subcellular localization, and sequence diversity of the Mlo family in plants. J Biol Chem 247(49):34993–35004
Kim MC, Panstruga R, Elliott C, Müller J, Devoto A, Yoon HW, Park HC, Cho MJ, Schulze-Lefert P (2002) Calmodulin interacts with MLO protein to regulate defence against mildew in barley. Nature 416(6879):447–451
Kim MC et al (2002) Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein. J Biol Chem 277(22):19304–19314
Bednarek P et al (2009) A glucosinolate metabolism pathway in living plant cells mediates broad-spectrum antifungal defense. Science 323(5910):101–106
Consonni C, Bednarek P, Humphry M, Francocci F, Ferrari S, Harzen A, Loren V, van Themaat E, Panstruga R (2010) Tryptophan-derived metabolites are required for antifungal defense in the Arabidopsis mlo2 mutant. Plant Physiol 152(3):1544–1564
Collins NC et al (2003) SNARE-protein-mediated disease resistance at the plant cell wall. Nature 425(30):973–977
Pajonk S, Kwon C, Clemens N, Panstruga R, Schulze-Lefert P (2008) Activity determinants and functional specialization of Arabidopsis PEN1 syntaxin in innate immunity. J Biol Chem 283(40):26974–26984
Bhat RA, Miklis M, Schmelzer E, Schulze-Lefert P, Panstruga R (2005) Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain. Proc Natl Acad Sci 102(8):3135–3140
Kwon C et al (2008) Co-option of a default secretory pathway for plant immune responses. Nature 451(7180):835–840
Kwaaitaal M, Keinath NF, Pajonk S, Biskup C, Panstruga R (2010) Combined bimolecular fluorescence complementation and förster resonance energy transfer reveals ternary SNARE complex formation in living plant cells. Plant Physiol 152(3):1135
Harwood WA (2012) Advances and remaining challenges in the transformation of barley and wheat. J Exp Bot 63(5):1791–1798
Lee W-S, Hammond-Kosack KE, Kanyuka K (2015) In planta transient expression systems for monocots. Recent Adv Gene Expr Enabling Technol Crop Plants:391–422
Nelson AJ, Bushnell WR (1997) Transient expression of anthocyanin genes in barley epidermal cells: potential for use in evaluation of disease. Transgenic Res 244:233–244
Hückelhoven R (2014) The effective papilla hypothesis. New Phytol 204(3):438–440
Stolzenburg MC, Aist JR, Israel HW (1984) The role of papillae in resistance to powdery mildew conditioned by the ml-o gene in barley. I correlative evidence. Physiol Plant Pathol 25(3):337–346
Both M, Csukai M, Stumpf MPH, Spanu PD (2005) Gene expression profiles of Blumeria graminis indicate dynamic changes to primary metabolism during development of an obligate biotrophic pathogen. Plant Cell 17(7):2107–2122
Panstruga R (2004) A golden shot: how ballistic single cell transformation boosts the molecular analysis of cereal – mildew interactions. Mol Plant Microbe Interact 5:141–148
Shirasu K, Nielsen K, Piffanelli P, Oliver R, Schulze-Lefert P (1999) Cell-autonomous complementation of mlo resistance using a biolistic transient expression system. Plant J 17(3):293–299
Acevedo-Garcia J, Spencer D, Thieron H, Reinstädler A, Hammond-Kosack K, Phillips AL, Panstruga R (2017) mlo-based powdery mildew resistance in hexaploid bread wheat generated by a non-transgenic TILLING approach. Plant Biotechnol J 15(3):367–378
Reinstädler A, Müller J, Czembor JH, Piffanelli P, Panstruga R (2010) Novel induced mlo mutant alleles in combination with site-directed mutagenesis reveal functionally important domains in the heptahelical barley Mlo protein. BMC Pant Biol 10:31
Elliott C, Müller J, Miklis M, Bhat RA, Schulze-Lefert P, Panstruga R (2005) Conserved extracellular cysteine residues and cytoplasmic loop-loop interplay are required for functionality of the heptahelical MLO protein. Biochem J 385(Pt 1):243–254
Elliott C, Zhou F, Spielmeyer W, Panstruga R, Schulze-Lefert P (2002) Functional conservation of wheat and rice Mlo orthologs in defense modulation to the powdery mildew fungus. Mol Plant-Microbe Interact 15(10):1069–1077
Ge X, Deng W, Lee ZZ, Lopez-Ruiz FJ, Schweizer P, Ellwood SR (2016) Tempered mlo broad-spectrum resistance to barley powdery mildew in an Ethiopian landrace. Sci Rep 6:29558
Panstruga R, Molina-Cano JL, Reinstädler A, Müller J (2005) Molecular characterization of mlo mutants in North American two- and six-rowed malting barley cultivars. Mol Plant Pathol 6(3):315–320
Hückelhoven R, Dechert C, Kogel KH (2003) Overexpression of barley BAX inhibitor 1 induces breakdown of mlo-mediated penetration resistance to Blumeria graminis. Proc Natl Acad Sci 100(9):5555–5560
Seeholzer S et al (2010) Diversity at the Mla powdery mildew resistance locus from cultivated barley reveals sites of positive selection. Mol Plant-Microbe Interact 23(4):497–509
Pliego C et al (2013) Host-induced gene silencing in barley powdery mildew reveals a class of ribonuclease-like effectors. Mol Plant-Microbe Interact 26(6):633–642
Panstruga R, Kim MC, Cho MJ, Schulze-Lefert P (2003) Testing the efficiency of dsRNAi constructs in vivo: a transient expression assay based on two fluorescent proteins. Mol Biol Rep 30(3):135–140
Nowara D, Schweizer P, Gay A, Lacomme C, Shaw J, Ridout C, Douchkov D, Hensel G, Kumlehn J (2010) HIGS: Host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell 22(9):3130–3141
Wahara M, Inoue C, Kohguchi T, Sugai K, Kobayashi K, Nishiguchi M, Yamaoka N, Yaeno T (2017) Improved method for in situ biolistic transformation to analyze barley–powdery mildew interactions. J Gen Plant Pathol 83(3):140–146
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Leissing, F., Reinstädler, A., Thieron, H., Panstruga, R. (2022). Gene Gun-Mediated Transient Gene Expression for Functional Studies in Plant Immunity. In: Kufer, T.A., Kaparakis-Liaskos, M. (eds) Effector-Triggered Immunity. Methods in Molecular Biology, vol 2523. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2449-4_5
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DOI: https://doi.org/10.1007/978-1-0716-2449-4_5
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