Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens

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 fixed^1,2,3or 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 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....

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 sheaths^6,8,36. 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.......

Materials

List of materials used in this article

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