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This manuscript details an optimized inoculation protocol that uses detached maize leaf sheaths for reproducible cytological, physiological, and molecular studies of maize interactions with fungal plant pathogens. The leaf sheaths facilitate real-time observation of cellular interactions between the living plant and fungus in unfixed tissues.
We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one originally applied to rice leaf sheaths and allows direct microscopic observation of fungal growth and development in living plant cells. Leaf sheaths collected from maize seedlings with two fully emerged leaf collars are inoculated with 20 µL drops of 5 x 105 spores/mL fungal spore suspensions and incubated in humidity chambers at 23 °C under continuous fluorescent light. After 24-72 h, excess tissue is removed with a razor blade to leave a single layer of epidermal cells, an optically clear sample that can be imaged directly without the necessity for chemical fixation or clearing. Plant and fungal cells remain alive for the duration of the experiment and interactions can be visualized in real-time. Sheaths can be stained or subjected to plasmolysis to study the developmental cytology and viability of host and pathogen cells during infection and colonization. Fungal strains transformed to express fluorescent proteins can be inoculated or co-inoculated on the sheaths for increased resolution and to facilitate the evaluation of competitive or synergistic interactions. Fungal strains expressing fluorescent fusion proteins can be used to track and quantify the production and targeting of these individual proteins in planta. Inoculated sheath tissues can be extracted to characterize nucleic acids, proteins, or metabolites. The use of these sheath assays has greatly advanced the detailed studies of the mechanisms of fungal pathogenicity in maize and also of fungal protein effectors and secondary metabolites contributing to pathogenicity.
Spatial and temporal analyses at the cellular level are critical for understanding the physiology and cytology of fungal-plant interactions. Foliar tissues that have been chemically fixed1,2,3or cleared and stained4, as well as artificial membranes5, have been used in the past to investigate the cytology of foliar pathogen development and plant-fungal interactions. However, investigation of infection events in living host tissues in real-time without fixation or clearing is challenging due to technical issues related to the prep....
NOTE: The workflow for the method is shown in Figure 1.
Figure 1: Steps in the optimized inoculation protocol using detached maize leaf sheaths. Spore suspension preparation, leaf sheath inoculation, and sample preparation for live-cell microscopy are highlighted in green (A
The examples below describe representative outcomes following the use of the maize leaf sheath inoculation method. These examples demonstrate the ease, speed, and precision with which observation and comparison of maize-fungus interactions can be accomplished in real-time with this optimized assay. Live-cell imaging also allows the extraction of quantitative information, providing a useful tool for comparative molecular, cytological, and physiological studies. Further details may be found in the original publications cit.......
The optimized leaf sheath inoculation method described here is modified from an original protocol that was developed for and has been applied to rice leaf sheaths6,8,36. It allows direct, detailed observations of fungal growth and development in living plant cells with either widefield or confocal microscopy. The protocol is suitable for characterization, comparison, and quantification of a variety of microscopic phenomena durin.......
The authors thank USDA-NIFA for their financial support (grant numbers 2018-67013-28489 and 2020-70410-32901). Any opinions, findings, conclusions, or recommendations expressed in this manuscript are solely those of the authors and do not necessarily reflect the views of the U.S. Department of Agriculture. We thank Science Without Borders visiting student from Brazil, Mayara de Silva, for the images that appear in Figure 6A and in Figure 7D. We also acknowledge the Department of Plant Pathology at the University of Kentucky for providing access to the Olympus confocal microscopes.
....Name | Company | Catalog Number | Comments |
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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