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Biology

用于真菌叶面玉米病原体感染的活细胞成像的分离玉米鞘

Published: September 15, 2023
doi:
10.3791/65755
, , , , ,
1Department of Plant Pathology,University of Kentucky, 2Department of Biological Sciences,University of Cincinnati, 3Bayer Crop Science, 4Everglades Research and Education Center,University of Florida

Summary

这份手稿详细介绍了一种优化的接种方案，该方案使用分离的玉米叶鞘对玉米与真菌植物病原体的相互作用进行可重复的细胞学、生理学和分子学研究。叶鞘有助于实时观察未固定组织中活植物和真菌之间的细胞相互作用。

Abstract

我们优化了一种方案，用半生物营养和坏死性叶面病原真菌接种玉米叶鞘。该方法从最初应用于水稻叶鞘的方法进行了修改，并允许直接在显微镜下观察活植物细胞中的真菌生长和发育。从具有两个完全出现的叶领的玉米幼苗收集的叶鞘接种20μL滴5×105 孢子/ mL真菌孢子悬浮液，并在连续荧光下在23°C的湿度室中孵育。24-72小时后，用剃须刀片去除多余的组织，留下单层表皮细胞，这是一种光学透明的样品，可以直接成像，无需化学固定或清除。植物和真菌细胞在实验期间保持活力，相互作用可以实时可视化。可以对鞘进行染色或进行等离子体分解，以研究感染和定植过程中宿主和病原体细胞的发育细胞学和活力。转化为表达荧光蛋白的真菌菌株可以接种或共接种在鞘上，以提高分辨率并促进竞争或协同相互作用的评估。表达荧光融合蛋白的真菌菌株可用于跟踪和量 化植物中这些单个蛋白的产生和靶向。可以提取接种的鞘组织以表征核酸、蛋白质或代谢物。这些鞘测定的使用极大地推进了对玉米真菌致病机制以及真菌蛋白效应物和次生代谢物的详细研究。

Introduction

细胞水平的空间和时间分析对于理解真菌-植物相互作用的生理学和细胞学至关重要。过去曾使用化学固定的叶面组织 1,2,3 或清除和染色 4 以及人造膜5 来研究叶面病原体发育和植物-真菌相互作用的细胞学。然而，由于与制备用于成像的光学透明样品相关的技术问题，在不固定或清除的情况下实时研究活体宿主组织中的感染事件具有挑战性。

1940 年代后期开发了一种分离的叶鞘接种方案，用于明场显微镜研究活水稻表皮细胞对稻瘟病真菌 Magnaporthe oryza6 的抗性。最近，通过将这种叶鞘方法的改良版本与表达荧光蛋白的真菌转化体相结合，以及高性能活细胞成像方案（包括落射荧光和共聚焦显微镜）7,8,9,10，11,12,13.

本文详细介绍了一种优化的接种方案，该方案使用分离的玉米叶鞘来观察半生物营养和坏死性叶面真菌病原体的感染过程。我们专门用它来研究炭疽病叶枯病和茎腐病的病原体禾本科菌（C. graminicola）和引起双枯病叶枯病和茎腐病的Stenocarpella maydis。但该方法应适用于其他半生物营养性和坏死性叶面真菌病原体。这些切除的叶鞘在感染和定植事件期间的细胞学和生理反应与整个叶片中的细胞学和生理反应相似12,14,15。此外，禾谷梭菌对鞘状表皮细胞的半生物营养定植类似于茎髓细胞的定植16,17。与叶片或茎髓组织相比，分离的鞘显示出更大的真菌渗透和定植的同步性和实验可重复性14,16,17,18。大多数玉米品种可用于该协议。然而，鞘中紫色颜料过多的近交系或杂交种不太合适，因为颜料会干扰成像。金禧甜玉米对我们的研究特别有用，因为未经处理的种子是市售的，植物极易受到许多叶面病害的影响，而且它们在温室中生长良好。1970 年代，美国炭疽病茎腐病的第一次流行导致印第安纳州甜玉米作物完全损失19,20。这种叶鞘接种方法可用于直接观察和量化活细胞与局部杀死的植物细胞中的真菌生长和发育，以证明对真菌感染的相容/不相容反应中的抗性反应，并实时测试同一鞘上的真菌菌株之间的相互作用。

Protocol

注：该方法的工作流程如 图1所示。 图1：使用分离的玉米叶鞘优化接种方案的步骤。 孢子悬浮液制备、叶鞘接种和活细胞显微镜样品制备分别以绿色 （A）、紫色 （B） 和橙色 （C） ?…

Representative Results

以下示例描述了使用玉米叶鞘接种方法后的代表性结果。这些示例表明，使用这种优化的检测方法可以实时完成玉米-真菌相互作用的观察和比较的便利性、速度和精确性。活细胞成像还可以提取定量信息，为比较分子、细胞学和生理学研究提供有用的工具。详情可参阅每份成功申请所引用的原始刊物。 示例数据1：在接种的叶鞘中使用染色和浆溶解来评估真菌状态和检?…

Discussion

这里描述的优化的叶鞘接种方法是从为水稻叶鞘6,8,36开发并已应用于水稻叶鞘的原始方案修改而来的。它允许使用宽场或共聚焦显微镜直接、详细地观察活植物细胞中的真菌生长和发育。该方案适用于玉米定植过程中各种微观现象的表征、比较和定量，包括相容与不相容相互作用期间的真菌发育和宿主反应;植物中特定?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢USDA-NIFA的财政支持（资助号2018-67013-28489和2020-70410-32901）。本手稿中表达的任何意见、发现、结论或建议仅代表作者的观点，并不一定反映美国农业部的观点。我们感谢来自巴西的无国界科学访问学生Mayara de Silva提供的图像，这些图像出现在图 6A 和 图7D中。我们还要感谢肯塔基大学植物病理学系提供奥林巴斯共聚焦显微镜。

Materials

Axiocam monochrome microscope camera ZEISS 426560-9010-000 Compatible with the Axioplan 2 microscope; provides low read noise and high speed for live cell imaging
Axioplan 2 epifluorescence microscope ZEISS N/A Allows live viewing and image/video capture of biological samples 
Benchtop centrifuge 24 X 1.5/2 mL Thermo Fisher Scientific 75002431 Sorvall Legend Micro 17; max speed: 13,300 rpm (17,000 x g)
Falcon bacteriological Petri dish with lid Fisher Scientific 08-757-105 Polystyrene material; hydrophobic surface
Filter paper  Fisher Scientific 09-920-115 Whatman grade 1 for Petri plate moist chambers
FV 3000 laser scanning confocal microscope Olympus N/A For visualization of fungal transformants' 
Germination paper Anchor Paper Co. SD7615L 76# heavy weight for plastic box moist chambers
Glass Petri dishes VWR International 75845-542 Type 1 class A, 33 expansion borosilicate glass;
complete set (cover + bottom), for Petri plate moist chambers
Glass wool  Ohio Valley Specialty Chemical  3350 For glass-wool filter units
Hemocytometer/Neubauer counting chamber and cover glass VWR International 15170-172 0.1 mm chamber depth; comes with two 0.4 mm cover glasses
Microscope coverslips Fisher Scientific 12-553-457  Borosilicate glass; 100/Pk.; 22 mm length, 22 mm width
Maize cultivar Golden Jubilee seeds West Coast Seeds Ltd., Delta, BC, Canada CN361 Matures in 95-105 days; seed type: F1
Microcentrifuge tubes  USA Scientific   1415-2500 1.5 mL capacity
Microscope slides  Fisher Scientific 12-550-123  Superfrost white tab slide; 76 mm length, 25 mm width
Oatmeal Agar (OA) VWR International 255210 Difco Oatmeal Agar, BD; 500 g
Nail polish Revlon 43671 Clear nail polish for sealing microscope slides; color 771 Clear
Non-skirted 96-well PCR plate USA Sientific 1402-9500 100 uL plate volume
Pestle for microcentrifuge tubes USA Scientific  1415-5390 Conical tip; polypropylene material
PlanApo 60X/1,00 WLSM water objective  Olympus 1-UB933 Compatible with the Olympus FV 3000 confocal microscope
Potato Dextrose Agar (PDA) VWR International 90000-758 Difco Potato Dextrose Media, BD; 500 g
Pro-Mix BX Premium Horticulture Supply Co. N/A Premium general-purpose growing medium formulated to provide
a balance of water retention and proper drainage
SC10 cone-tainers  Greenhouse Megastore  CN-SS-SC-10B 1.5 inch diameter, 8.25 inch depth, and a volume of 164 mL
SC10 cone-tainers tray Greenhouse Megastore  CN-SS-SCTR98 24 inch length x 12 inch width x 6.75 inch height; holds up to 98 of SC10 cone-tainers
Single edge razor blade Thermo Fisher Scientific 17-989-145 AccuTec blade; steel material; 38 mm length blade
Storage containers/boxes with latch closure Target 002-02-0405 Clear view storage boxes for rmoist chamber;
outside dimensions: 23 5/8 inch x 16 3/8 inch x 6 1/2 inch; 32 qt. capacity
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Tags

Keywords: MaizeLeaf SheathLive-cell ImagingFungal PathogensHemibiotrophicNecrotrophicPlant-pathogen InteractionsFungal ColonizationFungal PathogenicityMicroscopyFluorescent ProteinsReal-time VisualizationHost-pathogen InteractionsComparative Genomics
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Belisário, R., Torres, M. F., Buiate, E. A. S., Xavier, K. V., Nuckles, E. M., Vaillancourt, L. J. Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens. J. Vis. Exp. (199), e65755, doi:10.3791/65755 (2023).
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