Simplifying Barley Leaf Rust Research: An Easy and Reproducible Infection Protocol for Puccinia hordei on a Small Laboratory Scale
简化大麦叶锈病研究：一种简单且可重复的小型实验室规模的大麦锈病感染方案
(*contributed equally to this work, § Technical contact) 发布: 2023年07月20日第13卷第14期 DOI: 10.21769/BioProtoc.4721 浏览次数: 1221
评审: Zhibing LaiAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Feb 2022
Abstract
Barley (Hordeum vulgare) is one of the most important agricultural crops in the world, but pathogen infections regularly limit its annual yield. A major threat is the infection with the biotrophic leaf rust fungus, Puccinia hordei. Rust fungi have a complex life cycle, and existing resistances can be easily overcome. To address this problem, it is crucial to develop barley varieties with improved and durable resistance mechanisms. An essential step towards this goal is a simple and reproducible infection protocol to evaluate potential resistance phenotypes in the lab. However, available protocols sometimes lack detailed procedure or equipment information, use spore application methods that are not suitable for uniform spore dispersion, or require special mineral oils or engineered fluids. In addition, they are often optimized for pathogen-dedicated greenhouses or phytochambers, which may not be available to every research institute. Here, we describe an easy and user-friendly procedure to infect barley with Puccinia hordei on a small laboratory scale. This procedure utilizes inexpensive and simple tools to evenly split and apply spores to barley leaves. The treated plants are incubated in affordable and small phytocabinets. Our protocol enables a quick and reproducible infection of barley with leaf rust, a method that can easily be transferred to other rust fungi, including stripe rust, or to other plant species.
Key features
• Step-by-step infection protocol established for barley cv. Golden Promise, the gold standard genotype for genetic transformation
• Plant age–independent protocol
• Precise spore application by using inexpensive pipe cleaners for uniform symptom formation and increased reproducibility
• No specialized equipment needed
• Includes simple spore harvesting method
• Protocol is applicable to other biotrophic pathogens (stripe rust or powdery mildew) and other plants (e.g., wheat)
• Protocol is also applicable for a detached leaf assay
Graphical overview
Rust infection (锈病感染)
Biotrophic fungi (生物营养真菌)
Barley (大麦)
Golden Promise (Golden Promise)
Wheat germ agglutinin Alexa Fluor 488 staining (小麦胚芽凝集素Alexa Fluor 488染色)
Resistance assessment (抗性评估)
Background
In 2050, agriculture will have to feed more than nine billion people (FAO, 2009). This is an enormous challenge, especially in times of climate change, shortage of arable land, and spread of plant diseases. To secure future food supply, the development of high-yielding and stress-tolerant crops is urgently needed. Barley (Hordeum vulgare) is among the most important cereals in the world, needed as fodder and to brew beverages. However, infections with biotrophic fungal pathogens, such as rust or powdery mildew, severely threaten its annual yield, and existing resistance strategies can easily be overcome (König et al., 2012; Dinh et al., 2020). There is an ongoing arms race between pathogens and their host plants that necessitates the constant development of new and more durable resistance mechanisms. In the past decade, biotechnological breakthroughs such as the use of programmable DNA-binding proteins have revolutionized the precise targeting of genes to study their function (Miladinovic et al., 2021). In the future, this will allow the development of plants with potentially new resistance mechanisms. Consequently, reliable plant–pathogen infection protocols are a critical prerequisite to evaluate the infection phenotypes of the resulting plants in the lab. Working with biotrophic fungal pathogens can be particularly challenging because they require a living host and cannot be easily cultivated on plates. Here, we describe a comprehensive step-by-step protocol for reproducible rust infection assays in barley on a small lab scale. The protocol was established for Puccinia hordei infection of the barley cultivar Golden Promise (Skoppek et al., 2022). It includes a pre-propagation and easy harvesting protocol to produce a sufficient number of fresh spores for an infection trial. The detailed process information, uniform spore dispersal, and ease of handling allow a rapid establishment of the method. The equipment required is commonly available and inexpensive. In addition, we demonstrate that our infection protocol can easily be combined with macroscopic and microscopic evaluation methods to assess a certain resistance phenotype. Finally, the protocol can be easily transferred to other host plants and to other fungal pathogens, such as other rust fungi or powdery mildew.
Materials and reagents
Biological materials
Barley Golden Promise seeds
Urediniospores of leaf rust (Puccinia hordei; Ph) isolate I-80
Reagents and solutions for infection procedure
Tween 20 (Carl Roth, catalog number: 9127.1)
Sodium hypochlorite solution (NaClO + H2O) (Carl Roth, catalog number: 9062.3)
6% NaClO solution (see Recipe 1)
0.01% Tween 20 solution (see Recipe 2)
Optional: Reagents and solutions for fungal staining and microscopy
Wheat germ agglutinin, Alexa fluor 488TM conjugate (WGA AF488) (Thermo Fisher, catalog number: W11261)
Potassium hydroxide (KOH) (Carl Roth, catalog number: 6751.1)
Sodium chloride (NaCl) (Carl Roth, catalog number: 3957.2)
Potassium chloride (KCl) (AppliChem, catalog number: A1039.1000)
Disodium hydrogen orthophosphate (Na2HPO4) (Carl Roth, catalog number: 6751.1)
Potassium dihydrogen phosphate (KH2PO4) (Duchefa, catalog number: P0574.1000)
Acetic acid (CH3COOH) 100% (Carl Roth, catalog number: 3738.1)
Glycerol (C3H8O3) 86% (Carl Roth, catalog number: 4043.3)
Bleaching solution (see Recipe 3)
1 M KOH (see Recipe 4)
10× phosphate buffered saline (PBS) buffer pH 7.4 (see Recipe 5)
1× phosphate buffered saline (PBS) (see Recipe 6)
WGA AF488 stock solution (see Recipe 7)
WGA AF488 staining solution (see Recipe 8)
30% Glycerol solution (see Recipe 9)
Recipes
6% NaClO Solution
Reagent Final concentration Amount NaClO (12%) 6% 50 mL ddH2O n/a Ad 100 mL Caution: Dilute under fume hood, wear protective gear. Store at 4 °C in the dark (use brown bottle or wrap bottle in aluminum foil)
0.01% Tween 20 Solution
Reagent Final concentration Amount Tween 20 (100%) 0.01% 20 μL ddH2O n/a 199.98 mL Autoclave and store at room temperature (RT)
(Optional) Recipes for staining:
Bleaching solution
Reagent Final concentration Amount Ethanol (absolute) 75% 75 mL CH3COOH 100% 15% 15 mL ddH2O n/a Ad 100 mL Caution: Dilute under fume hood, wear protective gear. Store at RT
1 M KOH
Reagent Final concentration Amount KOH 1 M 5,611 g ddH2O n/a Ad 100 mL Store at RT.
10× PBS Buffer
Reagent Final concentration Amount NaCl 1.4 M 40.91g Na2HPO4·2H2O 10 mM 0.89 g KCl 27 mM 1.013 g KH2PO4 18 mM 1.23 g Dissolved in ddH2O n/a 500 mL Adjust to pH 7.4 with 10% KOH. For 1× PBS, dilute 10× PBS 1:10 with ddH2O (Recipe 6)
1× PBS Buffer
Reagent Final concentration Amount 10× PBS Buffer 1× 10 mL ddH2O n/a Ad 100 mL Store at RT.
WGA AF488 stock solution
Reagent Final concentration WGA AF488 1 mg/mL in H2O Prepare aliquots and store at -20 °C. Protect from light.
WGA AF488 staining solution
Reagent Final concentration Amount WGA AF488 stock solution 10 μg/mL in 1× PBS 10 μL/mL Always prepare fresh before use.
30% glycerol solution
Reagent Final concentration Amount Glycerol (86%) 30% 34,88 mL ddH2O n/a Ad 100 mL Store at RT
Laboratory supplies
Pots, 9–10 (maximum) cm diameter
Soil (Einheitserde classic; Profisubstrat, e.g., 814510; Meyer-Shop.de)
Metal trays (50 cm × 29.5 cm)
1.5 mL reaction tubes (Sarstedt, catalog number: 72.690.550)
Medical clay (Drug store)
Black ceramic tile (Hardware store)
Small spatula
Razor blade (Wilkinson)
Pipe cleaners, cotton, flexible, 6 mm diameter, 15–20 cm length (Carl Roth, catalog number: YC35.1 or Amazon e.g., VAUEN Cotton pipe cleaners)
Plastic cups (Plastikbecher.de, PP Becher 560/500 g natur A’50)
Parafilm (Carl Roth, catalog number: H666.1)
Lighter
Filtered tips 1 mL (Sarstedt, Biosphere plus, catalog number: 70.3050.255)
Silica beads (Carl Roth, catalog number: 1779.2)
Flexible tube for vacuum pump (0.8 cm diameter)
Scissor
Exsiccator with lid
Disposable gloves
Small plastic labels
2.0 mL reaction tubes (Sarstedt, catalog number: 72.695.500)
Object slides (Carl Roth, catalog number: 0656.1)
Cover slides (Carl Roth, catalog number: 1871.2)
Equipment
Precision balance (e.g., Sartorius)
Poly klima cabinet, true daylight (Poly klima GmbH, PK 520)
Vacuum pump (Welch, model: 2522C-02A)
Airbrush (GANZTON SP180K airbrush system, e.g., Amazon)
Water bath
4 °C refrigerator
-80 °C freezer
Scanner
Fluorescence microscope (Nikon, Nikon Ti Eclipse)
Software and datasets
NIS Element Software (Nikon)
Procedure
文章信息
版权信息
© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Skoppek, C. I. and Streubel, J. (2023). Simplifying Barley Leaf Rust Research: An Easy and Reproducible Infection Protocol for Puccinia hordei on a Small Laboratory Scale. Bio-protocol 13(14): e4721. DOI: 10.21769/BioProtoc.4721.
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分类
微生物学 > 微生物-宿主相互作用 > 真菌
植物科学 > 植物免疫 > 宿主-细菌相互作用
生物科学 > 微生物学
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