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.

NOTE: The workflow for the method is shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65755/65755fig01.jpg" /> 
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), purple (B), and orange (C) boxes, respectively. Created with BioRender.com. <a href="https://www.jove.com/files/ftp_upload/65755/65755fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Plant and fungal material
<ol>
	<li>Plant growth
	<ol>
		<li>Grow maize seedlings (see Table of Materials) in the greenhouse (14 h light/27 &#176;C and 10 h dark/22 &#176;C) in SC10 containers (see Table of Materials). Use a growth medium consisting of three parts commercial potting soil (see Table of Materials) and two parts steam-sterilized topsoil.</li>
		<li>Water seedlings for 2 min with an overhead irrigation system once every day, in the morning.</li>
		<li>Harvest seedlings at the V2 stage by cutting them at the soil level. The V2 growth stage is reached when seedlings are 5 to 10 cm tall and have two visible collared leaves21.</li>
		<li>Wrap the plants with a damp paper towel and place them in a plastic bag for transport to the laboratory for further processing.</li>
	</ol>
	</li>
	<li>Fungal cultures
	<ol>
		<li>Reactivate stored fungal silica stock cultures under aseptic conditions22by sprinkling approximately 50 granules of the infested silica on an appropriate agar medium. To avoid morphological changes and loss of pathogenicity, subculture strains no more than 3x after reactivation. Potato dextrose agar (PDA; see Table of Materials) is routinely used to culture C. graminicola while oatmeal agar (OA; see Table of Materials) is used for S. maydis.</li>
		<li>To prepare PDA for culturing fungal stocks, suspend 19.5 g of commercially prepared dehydrated PDA in 500 mL of deionized (DI) water. To make OA, boil 36&#160;g of commercially prepared dehydrated OA in DI water for 15-30 min, filter through three layers of cheesecloth, and bring filtrate to 500 mL with DI water. Autoclave media to sterilize.</li>
		<li>Augment the molten, cooled media with an appropriate antibiotic (e.g., hygromycin B, geneticin) when working with transformant strains. The final concentrations of hygromycin or geneticin in 500 mL of media are 250 &#181;g/mL and 100 &#181;g/mL, respectively.</li>
		<li>Incubate cultures under optimal conditions until a sporulating mycelium has developed. Colletotrichum graminicola and S. maydis grow well at 23 &#176;C with continuous fluorescent light and ambient humidity levels. Sporulation occurs by 7 days after inoculation (dai) for S. maydis or 14 dai for C. graminicola.</li>
	</ol>
	</li>
</ol>
2. Leaf sheath inoculations
<ol>
	<li>Glass-wool filter unit assembly
	<ol>
		<li>Use heavy-duty scissors or shears to cut off the cap and approximately 0.5 mm from the conical bottom of a 1.5 mL microcentrifuge tube (see Table of Materials).</li>
		<li>Place a piece of glass wool (approximately 0.5 cm x 0.5 cm; see Table of Materials) inside the cut tube to cover the center hole, as depicted in Figure 2.</li>
		<li>Wear gloves while cutting glass wool, as borosilicate glass can cause irritation.</li>
	</ol>
	</li>
	<li>Preparation of leaf sheath support racks
	<ol>
		<li>Cut a non-skirted 96-well PCR plate (see the Table of Materials) into six two-column (8 x 2 wells) support racks as depicted in Figure 3A.</li>
		<li>Flip the two-column racks so the conical bottoms of the wells face up and the flat tops face down (Figure 3B).</li>
		<li>Place each support in a glass Petri plate containing moistened filter paper and cover it with the lid (see Table of Materials). This unit functions as a small humidity chamber for the sheaths.</li>
	</ol>
	</li>
	<li>Inoculum preparation
	<ol>
		<li>For each fungal strain, place one assembled glass-wool filter unit into an intact sterile 1.5 mL microcentrifuge tube as depicted in Figure 2. The latter functions as a collection tube.</li>
		<li>Label the tubes.</li>
		<li>Harvest conidia from sporulating cultures by first flooding each plate with 3-4 mL of sterile DI water. More water may be necessary in some cases to produce a layer of liquid on the colony surface. Wetting agents are not necessary in this system.</li>
		<li>Loosen the spores from the agar by using a sterile conical-tip pestle (see Table of Materials) to scrape evenly across the entire plate.</li>
		<li>Apply 1 mL of the spore suspension to a glass wool filter unit aseptically and allow the spores to flow through into the collection tube by gravity.</li>
		<li>Centrifuge the collection tubes containing the filtered spore suspensions at 3,500 x g for 5 min. The spores should be pelleted at the bottom of the microcentrifuge tube. 
		NOTE: Centrifuging C. graminicola spores at higher speeds than recommended here results in a significant loss of viability.</li>
		<li>Pour off the liquid into an autoclavable container, add 1 mL of sterile DI water, and gently agitate to resuspend the pelleted spores. Centrifuge as in step 2.3.6.</li>
		<li>Wash spores 3x to remove any conidial matrix that may contain auto inhibitors that might reduce germination or penetration.</li>
		<li>After the third wash, add 300-500 &#181;L of sterile DI water to resuspend spores for quantification.</li>
		<li>Use a hemocytometer under a compound microscope at 100x magnification to determine the spore concentration. Spore staining is not necessary before counting.</li>
		<li>Prepare a 5 x 105 spores/mL suspension with sterile DI water. 
		NOTE: C. graminicola spore suspension can be kept at room temperature for no longer than 4 h before rapid loss of viability. Refrigerating conidia does not increase viability.</li>
	</ol>
	</li>
	<li>Sheath inoculations
	<ol>
		<li>Check relevant biosafety practices and procedures before inoculating plants with fungal strains.</li>
		<li>Remove the sheath from the first true leaf of V2 seedlings by running a thumbnail along the overlapping margin of the sheath, and gently loosen it from the shoot. Loosen the sheath from both sides of the shoot before trying to remove it.</li>
		<li>Cut the recovered leaf sheaths into 3-5 cm segments. It is not necessary to surface disinfect the sheaths before inoculation.</li>
		<li>Unroll each segment very gently to expose the inner (adaxial) epidermal layer.</li>
		<li>While preparing sheaths for inoculation, keep the remaining excised sheaths wrapped with a damp paper towel to avoid desiccation.</li>
		<li>Place 20 &#181;L of the fungal spore suspension on the inner surface at the center of the sheath piece, directly above the midrib, as depicted in Figure 4.</li>
		<li>Place the inoculated leaf sheaths horizontally, with the midrib placed at the bottom, in the support inside a glass Petri plate containing a moistened filter paper as depicted in Figure 5.</li>
		<li>Each rack holds up to seven sheaths. Inoculate at least five sheaths per strain to compensate for loss of replicates that may occur during sample preparation or incubation.</li>
		<li>Place the small humidity chambers into a clear storage box lined with moistened germination paper (see Table of Materials).</li>
		<li>Cover the box with the lid. Incubate the box at 23 &#176;C with continuous illumination for the intended time course, which depends on the fungus and on the fungal development stages that will be observed. A summary of the time course for maize sheaths inoculated with C. graminicola is provided in Table 1.</li>
		<li>Check every day for signs/symptoms of disease on the sheaths and keep both germination and filter papers moist. Leaf sheaths can be retained for up to 6 days without obvious plant cell death or degradation in the absence of inoculation.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65755/65755fig02.jpg" /> 
Figure 2: Glass-wool filter unit preparation. (A) A 0.5 cm x 0.5 cm glass-wool ball is placed inside microcentrifuge tube 1 that has its conical bottom removed. (B-C) The filter tube is then placed into microcentrifuge tube 2 to generate an assembled filter unit for spore suspension preparation. Created with BioRender.com. <a href="https://www.jove.com/files/ftp_upload/65755/65755fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65755/65755fig03.jpg" /> 
Figure 3: Method of cutting a non-skirted 96-well PCR plate. (A) PCR plate cut into six support racks, 8 x 2 wells. An example of a single sheath support is depicted in (B). Leaf sheaths are laid horizontally on the support. Created with BioRender.com. <a href="https://www.jove.com/files/ftp_upload/65755/65755fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65755/65755fig04.jpg" /> 
Figure 4: Sheath inoculation method. Single drop of inoculum directly applied to the adaxial surface of the sheath section. <a href="https://www.jove.com/files/ftp_upload/65755/65755fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65755/65755fig05.jpg" /> 
Figure 5: Sheath incubation method. Inoculated leaf sheaths placed horizontally in a support rack inside a glass Petri plate containing moistened filter paper. <a href="https://www.jove.com/files/ftp_upload/65755/65755fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Live-cell microscopy
<ol>
	<li>Sample preparation for microscopy
	<ol>
		<li>Rinse sheath pieces gently with sterile DI water to remove any unadhered spores or superficial fungal growth.</li>
		<li>Place the sheath on a clean glass microscope slide (see Table of Materials) with the adaxial (inside) surface of the sheath piece facing down and the abaxial (outside) surface uppermost.</li>
		<li>Use a new single-edge razor blade (see Table of Materials) to trim approximately 1 cm off the sheath ends and remove most of the lamina tissue on either side of the midrib.</li>
		<li>Hold the razor blade at an angle of 90&#176; relative to the sheath to shave tissue from the abaxial midrib and expose the epidermal layer on the adaxial surface above the inoculated spot. Try to maintain a uniform thickness. Once the leaf sheaths are shaved, further trimming is not recommended as this may damage the sample.</li>
		<li>Ensure that the shaved sections are no more than 1 cm x 1 cm in size. Avoid pressing on the center of the sheath during this process. When working with sheaths inoculated with different spore suspensions, disinfect gloves and change the razor blade between samples.</li>
		<li>Keep a clean glass microscope slide ready. Lift the sheath section from one edge and transfer it carefully to a new slide. The inoculated (adaxial) surface of the sheath should be uppermost, closest to the objective lens. Mount sections gently to avoid damage to fungal structures.</li>
		<li>Apply 60 &#181;L of sterile DI water to the section and add a 24 mm x 60 mm glass coverslip (see Table of Materials). Do this slowly to avoid air bubbles.</li>
		<li>When ready for microscopy, seal the coverslip with clear nail polish (see Table of Materials) by placing a small drop on each edge and then connecting the drops together with a nail polish brush to make a seamless seal. 
		NOTE: This sheath protocol can be used with cytological stains, e.g., 4&#8242;,6-diamidino-2-phenylindole (DAPI), neutral red, or trypan blue, or for observation of cell plasmolysis to evaluate plant cell viability. For sheath staining or cell plasmolysis assays, do not seal the coverslip.</li>
		<li>For plasmolysis assays, add a hypertonic solution (0.75 M sucrose or 1 M sodium chloride) to one edge of the coverslip and use a folded lint-free tissue paper to draw the solution across the sample to the other edge of the coverslip. For staining, use a similar method to apply the stain solution.</li>
	</ol>
	</li>
	<li>Widefield microscopy
	<ol>
		<li>Once the leaf sheaths are mounted on microscope slides, inspect them for fungal colonization using a widefield light microscope at 400x magnification.</li>
		<li>To observe fungal structures in detail, use a water immersion objective rather than oil for better image quality of the samples. Spherical aberration due to a refractive-index mismatch may cause image degradation. Water objectives are available for widefield light&#160;microscopes as well as confocal microscopes.</li>
		<li>To determine the relative amount of colonization per sheath, count the number of maize cells invaded from each fungal penetration site using a widefield light microscope at 100x magnification. This quantification is an appropriate screening step prior to any statistical analyses comparing strains, treatments, and/or developmental stages.</li>
	</ol>
	</li>
	<li>Confocal laser scanning microscopy
	<ol>
		<li>To observe fluorescent proteins in maize tissues during development of transgenic fungal strains, adjust the basic image acquisition parameters. Identify the best excitation/emission settings based on the selected fluorescent marker. 
		NOTE: A 60x water objective is preferable when analyzing fluorescent fusions constructed with secreted proteins. Modern confocal microscopes have highly corrected water immersion objectives to avoid artifacts and shape aberrations.</li>
		<li>If live-cell imaging of fluorescent proteins is intended, use untransformed fungal strains as controls for autofluorescence. Use the following image acquisition parameters to capture fungal-host dynamics on an inverted confocal microscope and for mCherry fluorescent protein. Set laser power up to 5%, 450-600 high voltage (HV) depending on the level of expression, 1.375 X gain, 3% offset, an optical zoom factor of 1 for the analysis of up to five maize cells per section, and optical zoom factor of 2-5 for individual hyphae. 
		NOTE: High-resolution Z-stack confocal images provide three-dimensional data and a better view of real-time interactions between the host and the pathogen.</li>
		<li>For quantification of fluorescence intensities corresponding to secreted fusion proteins, use image analysis software from the confocal manufacturer or an image processing program such as ImageJ, which can be freely downloaded online. Consult a manual for details on how to quantify fluorescent signals accordingly.</li>
	</ol>
	</li>
</ol>

Visualization of Cellular Interactions Between the Maize Sheaths and Fungi

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Y., Zhu, J., Xie, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+yeast+organelles+with+fluorescent+protein+markers.">Visualizing yeast organelles with fluorescent protein markers.</a> JoVE (Journal of Visualized Experiments). 182, e63846 (2022).</li><li>Westermann, B., Neupert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria-targeted+green+fluorescent+proteins:+convenient+tools+for+the+study+of+organelle+biogenesis+in+Saccharomyces+cerevisiae.">Mitochondria-targeted green fluorescent proteins: convenient tools for the study of organelle biogenesis in Saccharomyces cerevisiae.</a> Yeast. 16 (15), 1421-1427 (2000).</li><li>Lee, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ER+chaperone+and+signaling+regulator+GRP78/BiP+as+a+monitor+of+endoplasmic+reticulum+stress.">The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress.</a> Methods. 35 (4), 373-381 (2005).</li><li>Krishnakumar, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+maize+database+resource+that+captures+tissue-specific+and+subcellular-localized+gene+expression,+via+fluorescent+tags+and+confocal+imaging+(Maize+Cell+Genomics+Database).">A maize database resource that captures tissue-specific and subcellular-localized gene expression, via fluorescent tags and confocal imaging (Maize Cell Genomics Database).</a> Plant and Cell Physiology. 56 (1), e12-e12 (2015).</li><li>Wu, Q., Luo, A., Zadrozny, T., Sylvester, A., Jackson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+marker+lines+in+maize:+generation+and+applications.">Fluorescent protein marker lines in maize: generation and applications.</a> International Journal of Developmental Biology. 57 (6-7-8), 535-543 (2013).</li><li>O'Connell, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifestyle+transitions+in+plant+pathogenic+Colletotrichum+fungi+deciphered+by+genome+and+transcriptome+analyses.">Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses.</a> Nature Genetics. 44 (9), 1060-1065 (2012).</li><li>Torres, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Colletotrichum+graminicola+mutant+deficient+in+the+establishment+of+biotrophy+reveals+early+transcriptional+events+in+the+maize+anthracnose+disease+interaction.">A Colletotrichum graminicola mutant deficient in the establishment of biotrophy reveals early transcriptional events in the maize anthracnose disease interaction.</a> BMC Genomics. 17 (202), 1-24 (2016).</li><li>Andrie, R. M., Martinez, J. P., Ciuffetti, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+ToxA+and+ToxB+promoter-driven+fluorescent+protein+expression+vectors+for+use+in+filamentous+ascomycetes.">Development of ToxA and ToxB promoter-driven fluorescent protein expression vectors for use in filamentous ascomycetes.</a> Mycologia. 97 (5), 1152-1161 (2005).</li><li>Gordon, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucoamylase::+green+fluorescent+protein+fusions+to+monitor+protein+secretion+in+Aspergillus+niger.">Glucoamylase:: green fluorescent protein fusions to monitor protein secretion in Aspergillus niger.</a> Microbiology. 146 (2), 415-426 (2000).</li><li>Koga, H., Dohi, K., Nakayachi, O., Mori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+inoculation+method+of+Magnaporthe+grisea+for+cytological+observation+of+the+infection+process+using+intact+leaf+sheaths+of+rice+plants.">A novel inoculation method of Magnaporthe grisea for cytological observation of the infection process using intact leaf sheaths of rice plants.</a> Physiological and Molecular Plant Pathology. 64 (2), 67-72 (2004).</li><li>Xavier, K. V., Pfeiffer, T., Parreira, D. F., Chopra, S., Vaillancourt, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggressiveness+of+Colletotrichum+sublineola+strains+from+Sorghum+bicolor+and+S.+halepense+to+sweet+sorghum+variety+Sugar+Drip,+and+their+impact+on+yield.">Aggressiveness of Colletotrichum sublineola strains from Sorghum bicolor and S. halepense to sweet sorghum variety Sugar Drip, and their impact on yield.</a> Plant Disease. 101 (9), 1578-1587 (2017).</li></ol>

The authors declare that they have no competing financial interests and nothing to disclose.

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...

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 preparation of optically transparent samples for imaging.
A detached leaf sheath inoculation protocol was developed in the late 1940s for bright field microscopic investigation of resistance of living rice epidermal cells to the rice blast fungus Magnaporthe oryza6. More recently, detailed molecular, physiological, and cytological observations of host colonization by Colletotrichum and Magnaporthe species have been greatly facilitated by combining modified versions of this leaf sheath method with fungal transformants expressing fluorescent proteins, and high-performance live-cell imaging protocols, including epifluorescence and confocal microscopy7,8,9,10,11,12,13.
This paper details an optimized inoculation protocol using detached maize leaf sheaths for observation of infection processes by hemibiotrophic and necrotrophic foliar fungal pathogens. We have specifically used it to study Colletotrichum graminicola (C. graminicola), the causal agent of anthracnose leaf blight and stalk rot, and Stenocarpella maydis, which causes Diplodia leaf blight and stalk rot. However, the method should be applicable to other hemibiotrophic and necrotrophic foliar fungal pathogens. Cytological and physiological responses during infection and colonization events in these excised leaf sheaths are similar to those in entire leaf blades12,14,15. Furthermore, hemibiotrophic colonization of sheath epidermal cells by C. graminicola is similar to colonization of stalk pith cells16,17. Detached sheaths show greater synchronicity and experimental reproducibility of fungal penetration and colonization than leaf blades or stalk pith tissues14,16,17,18. Most maize varieties can be used for this protocol. However, inbreds or hybrids with excessive purple pigments in the sheaths are less suitable since the pigments interfere with imaging. Golden Jubilee sweet corn has been particularly useful for our studies because untreated seeds are commercially available, the plants are highly susceptible to many foliar diseases, and they grow well in the greenhouse. The first epidemics of anthracnose stalk rot in the United States resulted in the total loss of sweet corn crops in Indiana in the 1970s19,20. This leaf sheath inoculation method can be applied to directly observe and quantify fungal growth and development in living vs. locally killed plant cells, to demonstrate resistance reactions in compatible/incompatible responses to fungal infection, and to test interactions between fungal strains on the same sheath in real-time.

<table><tbody><tr><td>Axiocam monochrome microscope camera</td><td>ZEISS</td><td>426560-9010-000</td><td>Compatible with the Axioplan 2 microscope; provides low read noise and high speed for live cell imaging</td></tr><tr><td>Axioplan 2 epifluorescence microscope</td><td>ZEISS</td><td>N/A</td><td>Allows live viewing and image/video capture&amp;nbsp;of biological samples&amp;nbsp;</td></tr><tr><td>Benchtop centrifuge 24 X 1.5/2 mL</td><td>Thermo Fisher Scientific</td><td>75002431</td><td>Sorvall Legend Micro 17; max speed: 13,300 rpm (17,000 x g)</td></tr><tr><td>Falcon bacteriological Petri dish with lid</td><td>Fisher Scientific</td><td>08-757-105</td><td>Polystyrene material; hydrophobic surface</td></tr><tr><td>Filter paper&amp;nbsp;</td><td>Fisher Scientific</td><td>09-920-115</td><td>Whatman grade 1 for Petri plate moist chambers</td></tr><tr><td>FV 3000 laser scanning confocal microscope</td><td>Olympus</td><td>N/A</td><td>For visualization of fungal transformants&#039;&amp;nbsp;</td></tr><tr><td>Germination paper</td><td>Anchor Paper Co.</td><td>SD7615L</td><td>76# heavy weight for plastic box moist chambers</td></tr><tr><td>Glass Petri dishes</td><td>VWR International</td><td>75845-542</td><td>Type 1 class A, 33 expansion borosilicate glass;&lt;br/&gt; complete set (cover + bottom), for Petri plate moist chambers</td></tr><tr><td>Glass wool&amp;nbsp;</td><td>Ohio Valley Specialty Chemical&amp;nbsp;</td><td>3350</td><td>For glass-wool filter units</td></tr><tr><td>Hemocytometer/Neubauer counting chamber and cover glass</td><td>VWR International</td><td>15170-172</td><td>0.1 mm chamber depth; comes with two 0.4 mm cover glasses</td></tr><tr><td>Microscope coverslips</td><td>Fisher Scientific</td><td>12-553-457&amp;nbsp;</td><td>Borosilicate glass; 100/Pk.; 22 mm length, 22 mm width</td></tr><tr><td>Maize cultivar Golden Jubilee seeds</td><td>West Coast Seeds Ltd., Delta, BC, Canada</td><td>CN361</td><td>Matures&amp;nbsp;in 95-105 days; seed type: F1</td></tr><tr><td>Microcentrifuge tubes&amp;nbsp;</td><td>USA Scientific&amp;nbsp;&amp;nbsp;</td><td>1415-2500</td><td>1.5 mL capacity</td></tr><tr><td>Microscope slides&amp;nbsp;</td><td>Fisher Scientific</td><td>12-550-123&amp;nbsp;</td><td>Superfrost white tab slide; 76 mm length, 25 mm width</td></tr><tr><td>Oatmeal Agar (OA)</td><td>VWR International</td><td>255210</td><td>Difco Oatmeal Agar, BD; 500 g</td></tr><tr><td>Nail polish</td><td>Revlon</td><td>43671</td><td>Clear nail polish for sealing microscope slides; color 771 Clear</td></tr><tr><td>Non-skirted 96-well PCR plate</td><td>USA Sientific</td><td>1402-9500</td><td>100 uL plate volume</td></tr><tr><td>Pestle for microcentrifuge tubes</td><td>USA Scientific&amp;nbsp;</td><td>1415-5390</td><td>Conical tip; polypropylene material</td></tr><tr><td>PlanApo 60X/1,00 WLSM water objective&amp;nbsp;</td><td>Olympus</td><td>1-UB933</td><td>Compatible with the Olympus FV 3000 confocal microscope</td></tr><tr><td>Potato Dextrose Agar (PDA)</td><td>VWR International</td><td>90000-758</td><td>Difco Potato Dextrose Media, BD; 500 g</td></tr><tr><td>Pro-Mix BX</td><td>Premium Horticulture Supply Co.</td><td>N/A</td><td>Premium general-purpose growing medium formulated to provide&lt;br/&gt; a&amp;nbsp;balance of water retention and proper drainage</td></tr><tr><td>SC10 cone-tainers&amp;nbsp;</td><td>Greenhouse Megastore&amp;nbsp;</td><td>CN-SS-SC-10B</td><td>1.5 inch diameter, 8.25 inch depth, and a volume of 164 mL</td></tr><tr><td>SC10 cone-tainers tray</td><td>Greenhouse Megastore&amp;nbsp;</td><td>CN-SS-SCTR98</td><td>24 inch length x 12 inch width x 6.75 inch height; holds up to 98 of SC10 cone-tainers</td></tr><tr><td>Single edge razor blade</td><td>Thermo Fisher Scientific</td><td>17-989-145</td><td>AccuTec blade; steel material; 38 mm length blade</td></tr><tr><td>Storage containers/boxes with latch closure</td><td>Target</td><td>002-02-0405</td><td>Clear view storage boxes for rmoist chamber;&lt;br/&gt; outside dimensions: 23 5/8 inch x 16 3/8 inch x 6 1/2 inch; 32 qt. capacity</td></tr></tbody></table>

detached maize sheaths for live-cell imaging of infection by fungal foliar maize pathogens

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 &#181;L drops of 5 x 105 spores/mL fungal spore suspensions and incubated in humidity chambers at 23 &#176;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.

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...

Watch this Scientific Journal Video about Investigating Fungal Pathogenicity Mechanisms in Maize at JoVE.com

Author Spotlight: Investigating Fungal Pathogenicity Mechanisms in Maize 

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.

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

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one ...

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Research

JoVE Journal

Biology

1.4K Views.  University of Kentucky. Daarom wil ons team de pathogeniteitsmechanismen van schimmels in planten begrijpen om het ziektebeheer te verbeteren. We onderzoeken momenteel hoe de autochtone schimmel van maïs de eiwitsecretie reguleert buiten zijn huishoudelijke functie. Ons protocol toont een hoge synchroniciteit tussen replicaties en reproduceerbaarheid van schimmelkolonisatie.Met onze tests is het ook niet nodig om monsters te wissen of te fixeren voordat ze in realtime worden afgebeeld. De methode biedt een v...

Video: Auteur in de schijnwerpers: Onderzoek naar pathogeniteitsmechanismen van schimmels in maïs 

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast permitting in vivo phenotypic analysis. This method does not rely on maize transformation but exploits microbial genetics and secretory capabilities of pathogens. Herein, it allows inspection of in vivo delivered secreted proteins with high spatiotemporal resolution at different kinds of infection sites and tissues. The Trojan horse strategy can be utilized to transiently complement maize loss-of-function phenotypes, to functionally characterize protein domains, to analyze off-target protein effects, or to study onside protein overdosage, making it a powerful tool for protein studies in the maize crop system. This work contains a precise protocol on how to generate a Trojan horse strain followed by standardized infection protocols to apply this method to three different maize tissue types.

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

This work describes the cloning of an Ustilago maydis Trojan horse strain for the in situ delivery of secreted maize proteins into three different types of maize tissues.

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

Genetics

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Visualizing Early Infection Sites of Rice Blast Disease (Magnaporthe oryzae) on Barley (Hordeum vulgare) Using a Basic Microscope and a Smartphone

Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more sustainable strategies for plant health. Advances in methods that more effectively image plant-pathogen samples during infection and colonization have yielded tools such as the rice leaf sheath assay, which has been useful in monitoring infection and early colonization events between rice and the fungal pathogen, Magnaporthe oryzae. This hemi-biotrophic pathogen causes severe disease loss in rice and related monocots, including millet, rye, barley, and more recently, wheat. The leaf sheath assay, when performed correctly, yields an optically clear plant section, several layers thick, which allows researchers to perform live-cell imaging during pathogen attack or generate fixed samples stained for specific features. Detailed cellular investigations into the barley-M. oryzae interaction have lagged behind those of the rice host, in spite of the growing importance of this grain as a food source for animals and humans and as fermented beverages. Reported here is the development of a barley leaf sheath assay for intricate studies of M. oryzae interactions during the first 48 h post-inoculation. The leaf sheath assay, regardless of which species is being studied, is delicate; provided is a protocol that covers everything, from barley growth conditions and obtaining a leaf sheath, to inoculation, incubation, and imaging of the pathogen on plant leaves. This protocol can be optimized for high-throughput screening using something as simple as a smartphone for imaging purposes.

Visualizing Early Infection Sites of Rice Blast Disease Magnaporthe oryzae on Barley Hordeum vulgare Using a Basic Microscope and a Smartphone

This is a straightforward protocol of a barley leaf sheath assay using minimal reagents and common laboratory equipment (including a basic smartphone). The purpose is to visualize the early infection process of blast disease in labs without access to advanced microscopy equipment.

Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more ...

Developmental Biology

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro antifungal activity against a broad-spectrum of fungal pathogens and therefore have the potential to be used as antifungal agents in transgenic crops. In order to harness the full potential of plant defensins for diseases control, it is crucial to elucidate their mechanisms of action (MOA). With the advent of advanced microscopy techniques, live-cell imaging has become a powerful tool for understanding the dynamics of the antifungal MOA of plant defensins. Here, a confocal microscopy based live-cell imaging method is described using two fluorescently labeled plant defensins (MtDef4 and MtDef5) in combination with vital fluorescent dyes. This technique enables real-time visualization and analysis of the dynamic events of MtDef4 and MtDef5 internalization into fungal cells. Importantly, this assay generates a wealth of information including internalization kinetics, mode of entry and subcellular localization of these peptides. Along with other cell biological tools, these methods have provided critical insights into the dynamics and complexity of the MOA of these peptides. These tools can also be used to compare the MOA of these peptides against different fungi.

Plant defensins play an important role in plant defense against pathogens. For effective use of these antifungal peptides as antifungal agents, understanding their modes of action (MOA) is critical. Here, a live-cell imaging method is described to study critical aspects of the MOA of these peptides.

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro ...

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22)&#160;or&#160;phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.

An Agrobacterium-based injection (agroinjection) protocol is presented for the inoculation of foxtail mosaic virus&#160;and sugarcane mosaic virusclones into maize seedlings. Inoculation in this manner leads to viral infection, virus-induced gene silencing of marker genes, and viral overexpression of GFP.

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the ...

Improved Methods for Imaging Maize Leaf Primordia

Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging

In maize (Zea mays) and other grasses (Poaceae), the leaf primordia are deeply ensheathed and rolled within the leaf whorl, making it difficult to study early leaf development. Here, we describe methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging. The first method uses a wire stripper to remove the upper portions of older leaves, exposing the tip of the leaf primordium and allowing its measurement for more accurate transverse section sampling. The second method uses clear, double-sided nano tape to unroll and mount whole-leaf primordia for imaging. We show the utility of the two methods in visualizing and analyzing fluorescent protein reporters in maize. These methods provide a solution to the challenges presented by the distinctive morphology of maize leaf primordia and will be useful for visualizing and quantifying leaf anatomical and developmental traits in maize and other grass species.

Author Spotlight: Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging  

Maize leaf primordia are deeply ensheathed and rolled, making them difficult to study. Here, we present methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging.

In maize (Zea mays) and other grasses (Poaceae), the leaf primordia are deeply ensheathed and rolled within the leaf whorl, making it difficult to study ...

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

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