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

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 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 microscopy^{7,8,9,10,11,12,13}.

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 blades^12,14,15. Furthermore, hemibiotrophic colonization of sheath epidermal cells by C. graminicola is similar to colonization of stalk pith cells^16,17. Detached sheaths show greater synchronicity and experimental reproducibility of fungal penetration and colonization than leaf blades or stalk pith tissues^14,16,17,18. 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 1970s^19,20. 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.

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), purple (B), and orange (C) boxes, respectively. Created with BioRender.com. Please click here to view a larger version of this figure.

1. Plant and fungal material

1. Plant growth
   1. Grow maize seedlings (see Table of Materials) in the greenhouse (14 h light/27 °C and 10 h dark/22 °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.
   2. Water seedlings for 2 min with an overhead irrigation system once every day, in the morning.
   3. 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.
   4. Wrap the plants with a damp paper towel and place them in a plastic bag for transport to the laboratory for further processing.
2. Fungal cultures
   1. 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.
   2. 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 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.
   3. 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 µg/mL and 100 µg/mL, respectively.
   4. Incubate cultures under optimal conditions until a sporulating mycelium has developed. Colletotrichum graminicola and S. maydis grow well at 23 °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.

2. Leaf sheath inoculations

1. Glass-wool filter unit assembly
   1. 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).
   2. 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.
   3. Wear gloves while cutting glass wool, as borosilicate glass can cause irritation.
2. Preparation of leaf sheath support racks
   1. 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.
   2. Flip the two-column racks so the conical bottoms of the wells face up and the flat tops face down (Figure 3B).
   3. 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.
3. Inoculum preparation
   1. 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.
   2. Label the tubes.
   3. 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.
   4. Loosen the spores from the agar by using a sterile conical-tip pestle (see Table of Materials) to scrape evenly across the entire plate.
   5. 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.
   6. 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.
   7. 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.
   8. Wash spores 3x to remove any conidial matrix that may contain auto inhibitors that might reduce germination or penetration.
   9. After the third wash, add 300-500 µL of sterile DI water to resuspend spores for quantification.
   10. Use a hemocytometer under a compound microscope at 100x magnification to determine the spore concentration. Spore staining is not necessary before counting.
   11. 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.
4. Sheath inoculations
   1. Check relevant biosafety practices and procedures before inoculating plants with fungal strains.
   2. 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.
   3. Cut the recovered leaf sheaths into 3-5 cm segments. It is not necessary to surface disinfect the sheaths before inoculation.
   4. Unroll each segment very gently to expose the inner (adaxial) epidermal layer.
   5. While preparing sheaths for inoculation, keep the remaining excised sheaths wrapped with a damp paper towel to avoid desiccation.
   6. Place 20 µL 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.
   7. 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.
   8. 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.
   9. Place the small humidity chambers into a clear storage box lined with moistened germination paper (see Table of Materials).
   10. Cover the box with the lid. Incubate the box at 23 °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.
   11. 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.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Figure 4: Sheath inoculation method. Single drop of inoculum directly applied to the adaxial surface of the sheath section. Please click here to view a larger version of this figure.

Figure 5: Sheath incubation method. Inoculated leaf sheaths placed horizontally in a support rack inside a glass Petri plate containing moistened filter paper. Please click here to view a larger version of this figure.

3. Live-cell microscopy

1. Sample preparation for microscopy
   1. Rinse sheath pieces gently with sterile DI water to remove any unadhered spores or superficial fungal growth.
   2. 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.
   3. 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.
   4. Hold the razor blade at an angle of 90° 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.
   5. 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.
   6. 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.
   7. Apply 60 µL 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.
   8. 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′,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.
   9. 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.
2. Widefield microscopy
   1. Once the leaf sheaths are mounted on microscope slides, inspect them for fungal colonization using a widefield light microscope at 400x magnification.
   2. 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 microscopes as well as confocal microscopes.
   3. 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.
3. Confocal laser scanning microscopy
   1. 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.
   2. 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.
   3. 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.

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 cited for each successful application.

Example data 1: Use of staining and plasmolysis in inoculated leaf sheaths for evaluation of fungal status and detection of plant responses to fungal infection
Maize leaf sheaths have been used to visualize and compare the infection processes of Colletotrichum graminicola and Stenocarpella maydis in living host cells with bright field microscopy, allowing detailed descriptions of colonization patterns and time courses (unpublished results; Figure 6).

These studies revealed that S. maydis rapidly invades epidermal cells directly via undifferentiated hyphal swellings, unlike C. graminicola which produces melanized appressoria. Once inside the epidermal cells, both pathogens proceed from cell to cell by passing through apparently intact cell walls (Figure 6A,B).

Sheaths can be stained with trypan blue or DAPI to evaluate the nature and extent of colonizing fungal hyphae more easily (Figure 6). Trypan blue staining reveals that C. graminicola initially invades via thick primary hyphae that move between cells through very narrow connections. Later, the fungus switches to a necrotrophic stage, producing thinner secondary hyphae in the center of the colony (Figure 6 C-E)^12,14,16,17.

Figure 6: Unstained or stained inoculated maize leaf sheaths imaged with bright field or epifluorescence microscopy. (A) Intracellular hyphae 48 h post-inoculation (hpi) with Stenocarpella maydis. Infection occurs via slightly swollen hyphal tips (arrow). (B) Intracellular hyphae 48 hpi with Colletotrichum graminicola. Infection occurs via melanized appressoria (arrows). (C) Hyphae of C. graminicola, stained with trypan blue, progressing cell-to-cell at the advancing colony edge via narrow connections (arrow), 72 hpi. (D) Closer view of trypan-blue stained hypha of C. graminicola at 72 hpi, passing through the maize cell wall via a narrow connection (arrow). (E) Hyphae of C. graminicola, stained with trypan blue, in the colony center 72 hpi. Narrower secondary hyphae can be seen emerging from the thicker primary hyphae (arrows). (F) Hyphae of C. graminicola 72 hpi, at the advancing colony edge on a leaf sheath stained with DAPI. The stained fungal and plant nuclei fluoresce under UV light. Scale bars in each panel equal to 50 µm. Micrographs obtained using a monochromatic digital camera coupled with an epifluorescence microscope. Please click here to view a larger version of this figure.

Sheaths can be stained with neutral red and/or subjected to plasmolysis to study the developmental cytology and viability of maize cells during infection and colonization by pathogenic fungi (Figure 7).

Colletotrichum graminicola is a hemibiotrophic fungus that invades living maize cells with thick primary hyphae that are separated from the host cytoplasm by a membrane (Figure 7A-C)^12,14,16,17. Stenocarpella maydis, on the other hand, is a necrotrophic fungus that produces a phytotoxin²³ and kills the epidermal cells (which become granulated and fail to plasmolyze) before it invades them (Figure 7D).

Maize defense against C. graminicola and other foliar pathogens involves the accumulation of reactive oxygen species (ROS), particularly hydrogen peroxide (H₂O₂)^12,15. To investigate the oxidative chemistry of plant-pathogen interactions, 3,3′-diaminobenzidine (DAB) can be used for cell staining²⁴. DAB is oxidized by H₂O₂, producing a dark brown precipitate that is visible in the maize leaf sheaths (Figure 7 E,F)^12,24. Production of the pigment is an indicator of an oxidative burst related to a host resistance response.

Figure 7: Stained and/or plasmolyzed inoculated maize leaf sheaths imaged with bright field microscopy. (A) Leaf sheath inoculated with C. graminicola 24 hpi and subjected to plasmolysis and staining with neutral red. Living maize cells beneath appressoria (arrows) plasmolyze and stain, demonstrating that the pathogen does not kill the cells prior to invasion. (B) Leaf sheath 48 hpi with C. graminicola, subjected to plasmolysis and staining with neutral red. Maize cells that are colonized by primary hyphae (arrow) are still alive, establishing that C. graminicola invades cells as a biotroph. (C) Leaf sheath 48 hpi with C. graminicola, showing hyphae moving cell-to-cell at the advancing edge of the colony. Sheaths have been plasmolyzed but not stained. Uncolonized cells that are adjacent to the colony edge (arrows) continue to plasmolyze. (D) Leaf sheath 24 hpi with S. maydis, prior to penetration. The cells beneath the germinated spore (arrow) appear granulated and fail to plasmolyze, indicating the S. maydis kills them prior to penetration and that it behaves as a necrotroph. (E) Leaf sheath of resistant maize inbred Mp305 24 hpi with C. graminicola and stained with DAB. Dark staining indicates a strong oxidative response of the single cell in the center of the image, while halos of brown pigment can be seen surrounding most of the individual appressoria, and granulation can be observed in the cells at the top of the photo. (F) A closer view of the halos of brown pigment accumulated around appressoria, and deposition of pigment in the maize cell walls nearby (arrows). Scale bars in each panel equal to 50 µm, except for (F) which is 20 µm. Micrographs obtained using a monochromatic digital camera coupled with an epifluorescence microscope. Please click here to view a larger version of this figure.

Example data 2: Live-cell imaging with fungi expressing fluorescent proteins
Fungal hyphae expressing cytoplasmic fluorescent proteins can be visualized at high resolution in live sheath cells without the need for any stains by using an epifluorescence or confocal microscope (Figure 8).

Fluorescent proteins can be targeted to various fungal organelles, e.g., the mitochondria, nuclei, or the endomembrane system, in order to track their locations and activities in the living fungal cells in planta (Figure 8C)^25,26,27,28 (unpublished results). Fluorescent proteins can also be driven by different fungal promoters to serve as reporters for various functions: for example, RFP driven by the BiP chaperone secretion stress response protein is shown (Figure 8D)²⁹ (unpublished results). The use of leaf sheaths enables the visualization of individually tagged fluorescent fusion proteins that are accumulated and secreted by fungal hyphae as they colonize host cells^8,9 (Figure 8E). Transgenic maize lines expressing fluorescent proteins targeted to various cellular compartments, e.g., nuclei or plasma membrane, are also available^30,31, and can be used for inoculations to evaluate the effects of infection on maize cellular structures e.g., nuclei. Use of these transgenic maize lines demonstrated that nuclei in uninvaded maize cells typically migrate to the cell wall that is nearest to cells that are already colonized by C. graminicola (unpublished results; Figure 8F).

Figure 8: Epifluorescence (A, B) or confocal (C, D, E, F) imaging of Colletotrichum graminicola expressing fluorescent proteins. (A) Unstained, plasmolyzed leaf sheath 48 hpi with a C. graminicola strain expressing mRFP cytoplasmically under the control of the ToxA promoter³⁴. Significant autofluorescence is also noticed in these leaf sheaths. (B) Unstained leaf sheath 48 hpi with a C. graminicola strain expressing SGFP cytoplasmically under the control of the ToxA promoter. (C)Colletotrichum graminicola expressing SGFP targeted to the endomembrane system with an HDEL anchor³⁵. (D) Unstained leaf sheath 24 hpi with C. graminicola transformed with mRFP under the control of the promoter for the C. graminicola homolog of the BiP chaperone. The red fluorescence in the primary hypha is an indicator of BiP activation in response to secretion stress. (E) Accumulation of arabinofuranosidase::mCherry fusion protein in the C. graminicola WT primary hypha which is crossing the maize leaf sheath cell wall at 44 hpi. (F) Leaf sheath of a transgenic maize line expressing YFP targeted to the plant nuclei^30,31, 48 hpi with a strain of C. graminicola expressing mRFP cytoplasmically under the control of the ToxA promoter. Scale bars in each panel are equal to 20 µm, except for panel A where it is equal to 50 µm. Micrographs were obtained using a monochromatic digital camera coupled with an epifluorescence microscope. Images in (C-F) were captured with a laser scanning confocal microscope. Please click here to view a larger version of this figure.

Example data 3: Use of maize leaf sheaths to examine the detailed cytology of compatible/incompatible responses and interactions among fungal strains
A non-pathogenic C. graminicola mutant strain (cpr1 MT) was compared to the wild-type (WT) strain in maize leaf sheaths (Figure 9A,B)¹².

Live-cell imaging of the strains labeled with cytoplasmic fluorescent proteins demonstrated that the mutant was specifically impaired in movement from cell to cell within the maize leaf sheaths. The sheath protocol enabled precise quantification that revealed that the cpr1 MT was confined to the first colonized cell 95% of the time (Figure 9C)¹². This was in marked contrast to the WT, in which less than 5% of the infections remained within the first colonized cell in the same time frame¹². Interestingly, the cpr1 MT was able to grow saprophytically beyond the first initially infected cell in locally freeze-killed sheaths, like the WT strain (Figure 9D). This indicated that the inability of the cpr1 MT to colonize was specifically related to an active response of the living maize cell.

The leaf sheath inoculation assay was used to test interactions between the GFP-expressing cpr1 MT and RFP-expressing WT strains by co-inoculating them on the same leaf sheath¹². When the strains were inoculated close together (no more than 8 maize cells apart), the cpr1 MT was able to grow normally as a biotroph from cell to cell (Figure 9E,F). Plasmolysis assays confirmed that the cpr1 MT fungus entered living cells¹². All these detailed observations were made possible by the sheath assays and implied the existence of a diffusible factor that induces compatibility produced by the WT strain of C. graminicola but lacking in the cpr1 MT¹².

Figure 9: Compatible and incompatible interactions involving WT and cpr1 MT strains of Colletotrichum graminicola. Use of different fluorescent proteins allowed the two strains to be distinguished when co-inoculated on maize leaf sheaths¹². (A) The WT C. graminicola strain expressing cytoplasmic mRFP in maize leaf sheaths at 48 hpi. (B) The C. graminicola cpr1 MT strain expressing SGFP in maize leaf sheaths at 72 hpi. The cpr1 MT only rarely (<5% of the time) colonizes beyond the first infected cell, even up to 6 days after inoculation. (C) A closer view of the C. graminicola cpr1 MT strain expressing SGFP at 72 dpi. In this case, it managed to cross the cell wall into the next cell, but then it stopped developing. (D) The C. graminicola cpr1 MT strain expressing SGFP, 48 hpi in killed sheath tissues. The cpr1 MT strain colonizes nonliving maize sheath cells rapidly, similar to the WT. (E) When the WT-mRFP C. graminicola and cpr1 MT-GFP C. graminicola strains are co-inoculated close together on maize leaf sheaths (48 hpi), the cpr1 MT-GFP strain regains the ability to progress through living maize sheath cells normally, as a biotroph. Biotrophic growth inside the living cells was confirmed by plasmolysis assays. (F) A closer view of the C. graminicola cpr1 MT strain expressing SGFP, growing from cell-to-cell when inoculated adjacent to the WT (no more than 8 maize cells apart). Scale bars are equal to 50 µm (A, B, and E) or 20 µm (C, D, and F). Micrographs were obtained using a monochromatic digital camera coupled with an epifluorescence microscope. Please click here to view a larger version of this figure.

Inoculated sheaths have also been used to produce tissues for RNA extraction for transcriptional activity analysis^32,33. The sheaths were ideal for this purpose since they could be inspected before extraction to monitor the infection process, thus ensuring sample uniformity across multiple replications. These studies allowed the identification of waves of differentially expressed genes between Colletotrichum lifestyle transition stages^32,33.

Example data 4: Results of suboptimal experiments
Unsatisfactory live-cell imaging may be a result of insufficient trimming of leaf sheaths, leading to overlapping epidermal cell layers and optically opaque samples (Figure 10).

Figure 10: Suboptimal experimental outcome due to insufficient trimming of leaf sheaths.(A) Example of multiple epidermal cell layers interfering with the examination of fungal development in vivo. (B) Example of overlapping cell layers affecting live-cell imaging. Scale bars equal to 20 µm. Micrographs were obtained using a monochromatic digital camera coupled with an epifluorescence microscope. Please click here to view a larger version of this figure.

Another potential suboptimal experiment outcome is an unadjusted inoculum concentration that may result in low (Figure 11A) or high numbers of spores: the latter may result in reduced germination or appressorial penetration (Figure 11B). The latter can also hamper the quantification of maize cells colonized at each fungal penetration site.

Figure 11: Suboptimal experiment outcome due to improperly adjusted inoculum concentration. A. Low C. graminicola spore suspension concentration inoculated on sheaths resulting in low numbers of fungal penetration sites. B. High C. graminicola inoculum concentration on leaf sheaths causing multiple appressoria in close proximity, and reduced host cell penetration. Scale bars equal to 20 µm. Micrographs were obtained using a monochromatic digital camera coupled with an epifluorescence microscope. Please click here to view a larger version of this figure.

Table 1: Colletotrichum graminicola infection time course: Summary of the time course of maize leaf sheaths inoculated with C. graminicola strain M1.001 based on fungal development milestones throughout lifestyle transition. Microscopic observations were performed every 12 h.

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 during maize colonization, including fungal development and host responses during compatible versus incompatible interactions; production and secretion of specific fungal proteins in planta; and cytology and biochemistry of common or novel pathogenicity factors produced during the maize-fungal interaction. We have used this method with hemibiotrophic and necrotrophic pathogens of maize. We have not attempted inoculations with biotrophic pathogens (e.g., rust fungi) but the sheaths, since they remain alive, would also be useful for that application. We have also applied this method to sorghum sheaths for investigations of the cytology of host and cultivar-specific resistance^7,37. For sorghum sheaths, the only adjustment to the protocol was to fill the entire sheath with spore suspension, rather than applying a single drop in the center. This was necessary due to the smaller diameter of the sorghum sheaths.

The use of these sheath assays has greatly advanced our detailed studies on the mechanisms of host colonization and fungal molecules that are critical for pathogenicity. For instance, application of the method demonstrated non-host recognition of C. sublineola by maize, and of C. graminicola by sorghum, including hypersensitive resistance responses in both cases⁷. Maize leaf sheaths were also used to test interactions between fungal strains at a distance on the same sheath. In this case, co-inoculation of a pathogenic WT and non-pathogenic cpr1 MT strain together on living sheaths demonstrated the induction of susceptibility of maize cells by the WT strain and provided evidence for a diffusible factor involved in pathogenicity¹². The sheaths allowed differentiation of the two strains expressing different fluorescent proteins, and quantification and statistical comparisons of the colonization process for the strains inoculated either alone or together.

This maize leaf sheath assay has several advantages over whole plant inoculations for live-cell imaging. First, the protocol provides superior imaging, without necessity for clearing or fixation, of pathogens interacting with living host cells in real-time. For instance, studies using maize leaf sheaths enabled the detection of a distinct switch from biotrophy to necrotrophy in hemibiotrophic C. graminicola and facilitated functional comparisons to a non-pathogenic mutant^7,12,16,17. Although excised rice leaf sheaths inoculated with M. oryzae reportedly can present symptoms that do not correspond to those observed in whole rice plants³⁶, the timing and appearance of colonization, tissue collapse, and lesion development in maize sheaths is similar to that in whole leaves^12,14. A second advantage of the sheath assay is that it shows greater synchronicity across replications and experimental reproducibility of fungal colonization^12,18. While whole plant inoculation can result in irregular levels of fungal penetration¹⁸, the more highly controlled conditions of the sheath inoculations provide more synchronicity and allow more precise investigation and quantification of interactions. This increased synchronicity facilitated the use of the sheaths in transcriptome analyses^32,33. Furthermore, the sheath protocol enabled the necessary speed of sample preparation for transcriptome analysis: trimming, rinsing, and screening each sheath took no more than 2 min before they were flash frozen for RNA extraction^32,33.

Critical steps for successful live-cell imaging of infection include: 1) Sheaths should be used immediately after cutting for experiments. Excised inoculated sheaths should not be retained for more than 6 days: if fungal colonization is heavy, they will degrade more quickly¹²; 2) Be careful to choose sheaths that are of the same developmental age to ensure more reproducible results; 3) Use a fresh spore suspension from cultures that have achieved maximum sporulation; 4) Use sharp razor blades for more efficient trimming to produce optically clear samples; 5) Minimize excessive and unnecessary manipulation of samples as this process can negatively impact live structures and image quality; 6) Check humidity chambers daily to ensure that filter paper is sufficiently moist; and 7) When imaging samples on a confocal microscope, keep acquisition settings fixed during the experiment to avoid introducing intensity changes or bias when comparing fluorescent signals across samples. Fluorescent proteins may photobleach if laser intensity and duration are too high, and they may give weaker signals if tissue sections are kept under a sealed coverslip for a long time. Preliminary experiments, including all appropriate controls, should be performed to standardize and optimize conditions, materials, and timelines for the most reproducible results.

Troubleshooting
Hand-trimming is insufficient to produce optically clear sheaths
It is possible that the razor blade has become dull. If this is the case, dispose of it properly and switch to a new single-edge razor blade. Use moderate pressure and keep the blade at an angle of 90° to the sheath. Scrape the sample gently, but consistently, until observation of a flat and translucent section. It is advisable to check sheaths under a compound microscope before proceeding to the confocal laser scanning microscope.

Inoculated maize sheath is extremely fragile
This occurs when the fungus has colonized most of the plant tissue. Consequently, the tissue is degraded and collapsing. When preparing samples that are already in the necrotrophic stage, gently press the section against a microscope slide, apply a drop of sterile DI water, and scrape the sample one-two times with a clean coverslip.

Low spore germination and penetration in vivo
This may be due to a high number of spores inhibiting germination or penetration. Make sure inoculum concentration is adjusted to 5 x 10⁵ spores/mL. If the issue persists, adjust the concentration to 5 x 10⁴ spores/mL. As a control for the in vivo experiment, place 50 µL of the same spore suspension onto a fresh PDA plate and spread it evenly. Check spore viability and germination daily.

Asynchronous fungal development stages across sheaths
Make sure maize plants are at the same growth stage and sheath sections were obtained from the same node. Also, ensure that spores are viable and fungal cultures are at the optimum stage (e.g., no longer than 2-week-old cultures for C. graminicola) for inoculum preparation.