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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

NOTE: The workflow for the method is shown in Figure 1.

figure-protocol-182
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

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Axiocam monochrome microscope cameraZEISS426560-9010-000Compatible with the Axioplan 2 microscope; provides low read noise and high speed for live cell imaging
Axioplan 2 epifluorescence microscopeZEISSN/AAllows live viewing and image/video capture of biological samples 
Benchtop centrifuge 24 X 1.5/2 mLThermo Fisher Scientific75002431Sorvall Legend Micro 17; max speed: 13,300 rpm (17,000 x g)
Falcon bacteriological Petri dish with lidFisher Scientific08-757-105Polystyrene material; hydrophobic surface
Filter paper Fisher Scientific09-920-115Whatman grade 1 for Petri plate moist chambers
FV 3000 laser scanning confocal microscopeOlympusN/AFor visualization of fungal transformants' 
Germination paperAnchor Paper Co.SD7615L76# heavy weight for plastic box moist chambers
Glass Petri dishesVWR International75845-542Type 1 class A, 33 expansion borosilicate glass;
complete set (cover + bottom), for Petri plate moist chambers
Glass wool Ohio Valley Specialty Chemical 3350For glass-wool filter units
Hemocytometer/Neubauer counting chamber and cover glassVWR International15170-1720.1 mm chamber depth; comes with two 0.4 mm cover glasses
Microscope coverslipsFisher Scientific12-553-457 Borosilicate glass; 100/Pk.; 22 mm length, 22 mm width
Maize cultivar Golden Jubilee seedsWest Coast Seeds Ltd., Delta, BC, CanadaCN361Matures in 95-105 days; seed type: F1
Microcentrifuge tubes USA Scientific  1415-25001.5 mL capacity
Microscope slides Fisher Scientific12-550-123 Superfrost white tab slide; 76 mm length, 25 mm width
Oatmeal Agar (OA)VWR International255210Difco Oatmeal Agar, BD; 500 g
Nail polishRevlon43671Clear nail polish for sealing microscope slides; color 771 Clear
Non-skirted 96-well PCR plateUSA Sientific1402-9500100 uL plate volume
Pestle for microcentrifuge tubesUSA Scientific 1415-5390Conical tip; polypropylene material
PlanApo 60X/1,00 WLSM water objective Olympus1-UB933Compatible with the Olympus FV 3000 confocal microscope
Potato Dextrose Agar (PDA)VWR International90000-758Difco Potato Dextrose Media, BD; 500 g
Pro-Mix BXPremium Horticulture Supply Co.N/APremium general-purpose growing medium formulated to provide
a balance of water retention and proper drainage
SC10 cone-tainers Greenhouse Megastore CN-SS-SC-10B1.5 inch diameter, 8.25 inch depth, and a volume of 164 mL
SC10 cone-tainers trayGreenhouse Megastore CN-SS-SCTR9824 inch length x 12 inch width x 6.75 inch height; holds up to 98 of SC10 cone-tainers
Single edge razor bladeThermo Fisher Scientific17-989-145AccuTec blade; steel material; 38 mm length blade
Storage containers/boxes with latch closureTarget002-02-0405Clear 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

References

  1. Cheng, Y., Yao, J., Zhang, H., Huang, L., Kang, Z. Cytological and molecular analysis of nonhost resistance in rice to wheat powdery mildew and leaf rust pathogens. Protoplasma. 252 (4), 1167-1179 (2015).
  2. Hickey, E. L., Coffey, M. D.

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