The authors wish to thank Dr. Hudson W. P. de Carvalho for the support to develop this work. The authors are also grateful to the Laboratory of Electron Microscopy &#39;&#39;Prof. Elliot Watanabe Kitajima&#39;&#39; for providing the light microscopy facility. The authors thank&#160;Dr. Fl&#225;via Rodrigues Alves Patr&#237;cio for supplying the plant material with lesions.

1. Preparation of the buffering solution and reagents
<ol>
	<li>Prepare 2 M cacodylate buffer by adding 4.28 g of sodium cacodylate to 100 mL of distilled water and adjust the pH to 7.25 with 0.2 N HCl.</li>
	<li>Prepare 100 mL of the Karnovsky fixative solution by mixing 10 mL of 25% aqueous glutaraldehyde, 10 mL of 10% aqueous formaldehyde, 25 mL of 2 M cacodylate buffer, and 0.5 mL of 0.5 M CaCl226. Make up the volume to 100 mL with distilled water. 
	NOTE: The solution can be kept in a refrigerator for 6 months. 
	CAUTION: The cacodylate buffering solution is toxic; therefore, handle the fixative solution in a fume hood or an open area. Avoid inhaling the solution vapors and wear gloves while handling.</li>
	<li>Prepare aqueous Hoagland nutrient solution by mixing the following: 3 mM Ca(NO3)2.4H2O, 2 mM NH4H2PO4, 5 mM KH2PO4, 2 mM MgSO4.7H2O, 9.07 mM MnSO4, 0.765 mM ZnSO4.7H2O, 46.4 mM H3BO3, 0.09 mM Na2MoO4.H2O, 0.01 mM CuSO4, and 36 mM FeSO4.7H2O as iron-EDTA (ethylenediamine tetraacetic acid)27.</li>
</ol>
2. Plant samples and fungus inoculation
NOTE: For experiments on leaves affected by coffee rust, five 2-month-old seedlings of Coffee arabica cv. Catua&#237; were grown and kept in a greenhouse at the Center of Nuclear Energy in Agriculture (CENA) of the University of S&#227;o Paulo, Piracicaba, S&#227;o Paulo State, Brazil.
<ol>
	<li>Grow coffee plants in 500 mL plastic pots filled with aqueous Hoagland nutrient solution (pH of ~5.5) for 4 months in a growth chamber kept at 27 &#177; 3 &#176;C with a 12 h photoperiod created by LED lamps at the photon flux of 250 &#181;mol photons s-1 m-2. Replace the Hoagland nutrient solution every week for 4 months.</li>
	<li>Inoculate four expanded leaves from five plants on their abaxial surfaces with 1 x 103&#160;H. vastatrix uredospores following the method described in reference28. After inoculation, keep the plants for 48 h in the dark by covering them with a black plastic bag. Harvest the lesions 30 days after inoculation.</li>
	<li>Harvest characteristic lesions caused by Cercospora coffeicola from Coffea arabica cv. Obat&#227; plants located (coordinates: -22.906506126269942, -47.015075902025266) at the Biological Institute, Campinas, S&#227;o Paulo State, Brazil. Before processing the sample, analyze the lesions in a stereomicroscope to verify the presence of coffee C. coffeicola conidia. Then, mount some slides with the conidia to confirm the disease etiology22.</li>
</ol>
3. Sample harvesting, fixation, and dehydration
<ol>
	<li>Using a scalpel and tweezer, harvest a ~10 mm2 leaf sample at the middle region of the lesion (yellow spots; Figure 1) and immerse it in 30 mL of Karnovsky fixative solution (Figure 1&#160;and Figure 2A). The fixation step can take place in a refrigerator for 48 h.</li>
	<li>For at least four times, subject the leaf sample to a low vacuum (500-600 mBar) using an oil pump for 15 min each to increase the permeability of the fixative solution in the leaf tissue. Perform this step with sample rotation (Figure 1).</li>
	<li>After fixation, wash the leaf sample three times in 0.5 M cacodylate buffer (pH 7.2) diluted in distilled water for 5 min each and then transfer it to a graded ethanolic series (30%, 50%, 70%, 90% (2x), and 100% (2x)) for 15 min at each ethanol concentration (Figure 1 and Figure 2B).</li>
</ol>
4. Historesin embedding procedure
<ol>
	<li>Gradually transfer the samples to glycol methacrylate (GMA) in three steps, following the manufacturer&#39;s instructions. First, make Solution A by mixing the GMA powder (1 g) with 100 mL of basic resin (historesin kit; Table of Materials) under magnetic agitation, and then follow the below steps.
	<ol>
		<li>Immerse the samples in 1:2 Solution A: 100% ethanol for 3 h.</li>
		<li>Immerse the samples in 1:1 Solution A: 100% ethanol for 3 h.</li>
		<li>Immerse the samples in a pure basic resin for 2-4 days. During this step, subject the samples to a low vacuum at least four times a day for 15 min followed by rotation.</li>
	</ol>
	</li>
</ol>
5. Polymerization
NOTE: The polymerization process requires 1.2 mL plastic molds, basic resin, and hardener (see Table of Materials for the details of the commercial kit).
<ol>
	<li>Mix 15 mL of Solution A (step 4.1) with 1 mL of the hardener in a beaker with rotation for 2 min to produce the polymerization solution (Solution B).</li>
	<li>Put 1.2 mL of the polymerization solution (Solution B) in plastic molds. Using a wooden pick, transfer the lesioned leaf samples from pure basic resin to Solution B (Figure 2C). Avoid using tweezers as they can cause tissue crushing.</li>
	<li>Ensure to quickly orient leaf samples perpendicular to the plastic molds as solution B quickly becomes viscous within 5 min. More than one lesioned leaf sample can be placed in a single mold. 
	NOTE: It is recommended to practice the above step several times before applying for many samples. When there are many samples, the polymerization time is different among the molds and the perpendicular orientation of the leaf samples may be difficult to achieve.</li>
	<li>When the perpendicular orientation of the leaf samples is achieved, wait for 30 min, and then transfer the plastic mold to a plastic or glass chamber containing silica gel to prevent humidity. Wait for 2-3 h for polymerization.</li>
	<li>Once the resin and the leaf sample are polymerized after the 2-3 h period, detach the resulting block from the plastic mold by sanding the block base with a sanding file. Then, glue the block to a piece of wood (Figure 2D).</li>
</ol>
6. Sectioning
<ol>
	<li>Using a rotative microtome equipped with 8 cm steel blades (Figure 2E), cut the block into 5 &#181;m thick sections. Place the sections onto glass slides covered with distilled water. Transfer the slides with the sections floating over water to a hot plate at 40 &#176;C to dry and promote the adhesion of the sections to the glass slides.</li>
	<li>After drying (Figure 2F), label the glass slides with the block reference name and the slide number.</li>
</ol>
7. Double staining process
<ol>
	<li>Cover the sections with 2 mL of 5% cotton blue in lactophenol (40% glycerol, 20% phenol, and 20% lactic acid in water) and heat them on a hot plate at 45 &#176;C for 5 min (Figure 3A).</li>
	<li>Remove the excess dye by washing the slide three times in a beaker filled with distilled water (Figure 3B-D).</li>
	<li>Stain with 2 mL of 0.01% ruthenium red in water for 1 min (Figure 3E).</li>
	<li>Remove the excess dye by washing the slide three times in a beaker filled with distilled water (Figure 3F,G).</li>
	<li>Put a drop of distilled water over the sections and cover the sections with a 24 mm x 60 mm coverslip for performing light microscopy analysis.</li>
</ol>

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<li>Luft, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ruthenium+red+and+violet.+1.+Chemistry%2C+purification%2C+methods+of+use+for+electron+microscopy+and+mechanism+of+action%5BTitle%5D)+AND+%22The+Anatomical+Record%22%5BJournal%5D)">Ruthenium red and violet. 1. Chemistry, purification, methods of use for electron microscopy and mechanism of action.</a> The Anatomical Record. 171 (3), 347-368 (1971).</li>
<li>Buckeridge, M. S., Cavalari, A. A., Silva, G. B. D. A. Cell Wall. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBuckeridge%2C+MS+%22Plant+Physiology%22">Plant Physiology</a>. Kerbauy, G. B. , Guanabara Koogan. Rio de Janeiro. in Portuguese 165-181 (2013).</li>
</ol>

The authors declare no conflicts of interest.

The present work introduces an alternative double-staining histochemical test to investigate the pectin composition of cell walls that encapsulates haustoria in a biotrophic pathosystem. The aim is also to demonstrate the efficacy of the method to detect necrotrophic fungus and cell wall changes induced by it. Here, pectin of coffee parenchyma cell walls can encapsulate both the neck and the haustorium of the rust fungus Hemileia vastatrix. Silva et al. have also described encapsulation by cellulose and callose in the coffee-H. vastatrix pathosystem14,29. Among the cell wall polymers associated with defense mechanisms, pectin plays an important role in the plant-pathogen system10,11,12. Therefore, knowledge of pectin functions is of great histopathological value.
Cercosporiosis in coffee, caused by Cercospora coffeicola, damages the leaf tissues and eventually leads to cell death. These symptoms are mainly caused by the activity of cercosporin and cell wall degrading enzymes20. Studies using ruthenium red demonstrated a loss of cell wall integrity, similar to a previous study on persimmon leaves infected by Pseudocercospora kaki30. The histopathological analysis using the double-staining method demonstrated the presence of the fungal hyphae and this analysis is effective to detect fungal hyphae in the intercellular spaces. Alternatively, scanning electron microscopy (SEM) has shown efficacy to observe C. coffeicola hyphae25; however, the availability of such sophisticated equipment, along with the laborious work of sample preparation, is a limiting factor. Moreover, the SEM analysis does not allow the chemical recognition of pectin in cell walls. Conversely, double staining is an important method to detect pectin changes. However, the method described here has a limitation with regards to the resolution of light microscopy that does not allow an increase in details of the ultrastructure of plant-fungal interaction. Also, the double staining method presented here is not specific to detect fungal species and additional molecular essays must, therefore, be conducted.
The sample preparation follows routine protocols in plant anatomy; however, some points need special attention. For instance, fixation is a critical step. The size of the samples and the care in harvesting them are essential for good fixation. Time, temperature, pH, and osmolarity are crucial for plant tissues31. The lesioned leaf tissues are aerial organs and must be subjected to a mild vacuum to improve fixation. The use of glycol methacrylate (GMA) requires ethanol as a dehydration solvent; tissues that are not properly dehydrated can present difficulties during the embedding process. This condition is worsened when the plant tissues have many phenolic compounds, as in the case of coffee leaves. Moreover, dealing with small parts of tissues is important; otherwise, alternative embedding methods are required32.
The double-staining technique presented here is an adaptation of the protocol reported by Marques et al.33. In that protocol, the authors used cotton blue (5%) and 1% safranin to distinguish fungal structures and plant cell walls. The technique was useful to detect the presence of fungus in different pathosystems, such as Colletotrichum acutatum-citrus petals and Guignardia citricarpa-citrus fruit33&#160;Plasmodiophora brassicae in Arabidopsis thaliana34, Elsino&#235; ampelina in Vitis labrusca35, and others. Recently, Marques and Soares36compiled a series of microscopical techniques, including the light and fluorescence methods, to distinguish the plant and fungal features37. However, in some regions or countries, such as Brazil, fluorescence microscopes and SEM may not be available; therefore, the use of light microscopes is an important and cheaper alternative for research and even didactic methods employed at universities and high schools. Fungal hyphae have been microscopically detected in both plant and animal tissues by cotton blue dye36,38,39,40. This is related to the positive reaction of the dye with the chitin-rich fungal wall40. The cotton blue formula includes lactophenol, which is a solution that acts as a mordant to the tissue41, thereby preserving the fungal structure when mixed with cotton blue.
Here, 0.05% ruthenium red was used to replace safranin. The positive aspect presented in this study is that pectin, a specific polymer of the cell wall, was stained to provide important qualitative data. Ruthenium red is a reagent used to detect acidic groups of pectin polyuronic acids42,43,44. It is specific to pectin and does not stain other carbohydrate components of the cell wall (i.e., cellulose or callose). Chains of galacturonic acids arranged in different structural and biochemical backbones compose pectin8,9,45. The reactivity of ruthenium red to the cell wall depends on the degree of methyl esterification11. The degree of methyl esterification depends on the activity of pectin methyl esterases (PME), and thus the ruthenium red was also used as a tool to discriminate the PME activity11.
Thus, the double-staining method described here is a useful tool to verify pectin modifications in plant-fungi interactions. To the best of the authors&#39; knowledge, this is the first report of pectin in the haustorial encapsulation of coffee rust fungi. Interestingly, the double-staining method was also important to observe fungal structures, to describe the damage caused to the pectin-rich cell wall, and to verify the fungal reproductive structures. Further histopathological studies on rusts, anthracnose, cercosporiosis, smuts and other biotrophic, hemibiotrophic, and necrotrophic plant-fungal interactions should be conducted to investigate the potential use of this technique in different pathosystems.

Cell wall defense mechanisms in plants are crucial to restrain fungal infection. Studies have reported changes in cell wall thickness and composition since the 19th century1,2. These changes can be induced by a fungal pathogen that stimulates the formation of a papilla, which prevents fungi from entering the cell or could be used to encapsulate the hyphae to isolate the host cell protoplast from the fungal haustoria. The production of a dynamic cell wall barrier (i.e., papillae and a fully encased haustorium) is important to promote plant resistance3. Histopathological studies on fungus-related diseases have investigated the occurrence of these mechanisms and have described the cell wall polymers, cellulose, hemicellulose (arabinoxylans), and callose as resistance mechanisms to fungal attack4,5,6,7.
The cell wall is the first barrier against microorganismal attack, impairing the plant-fungal interaction. Pectic polysaccharides compose the cell wall and account for about 30% of the cell wall composition in primary cells of eudicot plants in which homogalacturonans are the most abundant polymer (roughly 60%)8. The Golgi secretes complex pectin compounds that comprise the galacturonic acid chains, which may or may not be methylated8,9. Since 2012, the literature has pointed out that the degree of pectin methyl esterification is critical to determining the compatibility during infection by microbial pectic enzymes10,11,12. Thus, protocols are required to verify the presence and distribution of pectic compounds in plant-fungal pathosystems.
Various techniques have been used to detect the encapsulation of papillae or haustoria. The reference methods used are transmission electron microscopy (TEM) of fixed tissue and light microscopy of living and fixed tissues. Regarding TEM, several studies have demonstrated the structural role of cell wall appositions in fungal resistance13,14,15,16, and that the use of lectins and antibodies is an intricate method to locate carbohydrate polymers16. However, studies show that light microscopy is an important approach and that the histochemical and immunohistochemical tools allow a better understanding of the composition of papillae and haustorium encasement6,7.
Pathogenic fungi show two main types of lifestyles: biotrophic and necrotrophic. Biotrophic fungi depend on living cells for their nutrition whereas necrotrophic fungi kill the host cells, and then live in the dead tissues17. In Latin America, coffee leaf rust, caused by the fungus Hemileia vastatrix, is an important disease in coffee crops18,19. Hemileia vastatrix presents a biotrophic behavior and, among the structural changes observed in resistant coffee species or cultivars, a hypersensitivity response, deposition of callose, cellulose, and lignin on the cell walls, as well as cell hypertrophy14&#160;have been reported. To the authors&#39; knowledge, the literature does not report information on the importance of pectin in coffee rust resistance. On the other hand, necrotrophic fungi that cause cercosporiosis target pectin via a set of enzymes associated with cell wall degradation, such as pectinases and polygalacturonase20. Cercosporiosis in coffee, caused by the fungus Cercospora coffeicola is also a major threat to coffee crops21,22. This fungus causes necrotic lesions in both leaves and berries. After penetration, C. coffeicola colonizes plant tissues through intracellular and intercellular pathways23,24,25.
The present protocol investigates the presence of fungal structures and pectin on cell walls. This protocol is useful to identify the plant response associated with pectin (stained with ruthenium red dye, which is specific to acidic groups of polyuronic acids of pectin), induced by the host in a biotrophic interaction with fungus. It also helps to verify the effect of necrotrophic fungi on the degradation of pectic cell walls. The present results indicate that the double staining method is effective to discriminate structures and the reproductive phase of fungi.

<table><tbody><tr><td>Blades DB80 HS</td><td>Leica</td><td>14035838383</td><td>Sectioning</td></tr><tr><td>Cacodylate buffer</td><td>EMS</td><td># 11652</td><td>Fixation</td></tr><tr><td>Cotton Blue Lactophenol</td><td>Metaqu&amp;iacute;mica</td><td>70SOLSIG024629</td><td>Staining</td></tr><tr><td>Formaldehyde</td><td>EMS</td><td>#15712</td><td>Fixation</td></tr><tr><td>Glutaraldehyde</td><td>EMS</td><td>#16216</td><td>Fixation</td></tr><tr><td>Historesin Kit</td><td>Technovit /EMS</td><td>#14653</td><td>Historesin for embedding</td></tr><tr><td>Hot plate</td><td>Dubesser</td><td>SSCD25X30-110V</td><td>Staining</td></tr><tr><td>Microscopy</td><td>Zeiss</td><td>#490040-0030-000</td><td>Image capture</td></tr><tr><td>Microtome (Leica RM 2540)</td><td>Leica</td><td>149BIO000C1 14050238005</td><td>Sectioning</td></tr><tr><td>Plastic molding cup tray</td><td>EMS</td><td>10176-30</td><td>Staining</td></tr><tr><td>Ruthenium red</td><td>LABHouse</td><td>#006004</td><td>Staining</td></tr><tr><td>Software Axion Vision</td><td>Zeiss</td><td>#410130-0909-000</td><td>Image capture</td></tr><tr><td>Vaccum pump</td><td>Prismatec</td><td>131 TIPO 2 V.C.</td><td>Fixation</td></tr></tbody></table>

double-staining method to detect pectin in plant-fungus interaction

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an efficient inducible mechanism to isolate the fungal haustoria from the plant cell protoplast. Conversely, pectin, one of the polymeric components of the cell wall, is a target of several pectolytic enzymes in necrotrophic interactions. Here, a protocol to detect pectin and fungal hyphae through optical microscopy is presented. The pectin-rich encapsulation in the cells of coffee leaves infected by the rust fungus Hemileia vastatrix and the mesophyll cell wall modification induced by Cercospora coffeicola are investigated. Lesioned leaf samples were fixed with the Karnovsky solution, dehydrated, and embedded in glycol methacrylate for 2-4 days. All steps were followed by vacuum-pumping to remove air in the intercellular spaces and improve the embedding process. The embedded blocks were sectioned into 5-7 &#181;m thick sections, which were deposited on a glass slide covered with water and subsequently heated at 40 &#176;C for 30 min. Next, the slides were double-stained with 5% cotton blue in lactophenol to detect the fungus and 0.05% ruthenium red in water to detect pectin (acidic groups of polyuronic acids of pectin). Fungal haustoria of Hemileia vastatrix were found to be encapsulated by pectin. In coffee cercosporiosis, mesophyll cells exhibited dissolution of cell walls, and intercellular hyphae and conidiophores were observed. The method presented here is effective to detect a pectin-associated response in the plant-fungi interaction.

The cotton blue lactophenol staining on the GMA-embedded section revealed the presence of several fungal structures between and inside coffee mesophyll cells in both biotrophic and necrotrophic fungal interactions.
In the biotrophic pathosystem, when stained using the double-staining method, Hemileia vastatrix hyphae containing cell walls and the dense protoplast content appear in dark blue in both spongy and palisade parenchyma (Figure 4A,B). The haustorium mother cell (Hmc) and the haustoria also exhibit a strong dark blue color (Figure 4C). When counterstained with ruthenium red, the fungal distribution in the intercellular spaces is clearly defined (Figure 4D). The presence of a dark blue Hmc helps to detect the infection zone. During the interaction, H. vastatrix Hmc broke the host cell wall and developed a haustorial neck that could be surrounded by pectic compounds (Figure 4E,F). Nevertheless, the pectin-rich encapsulation (pink-red color) of the haustorial neck is not capable of preventing haustorial formation (Figure 4E,F). In some cases, the pectin-rich encapsulation encased the haustorium incompletely (Figure 4G) and in some cases, the haustorium is fully encapsulated (Figure 4H).
In the necrotrophic interaction, the double staining protocol was also useful to verify the interaction of Cercospora coffeicola with coffee mesophyll tissues. At the lesion border, where the fungus is not present, the pectin-rich cell wall kept its integrity (Figure 5A). The double staining method demonstrated the presence of intercellular hyphae (Figure 5B,C). In interaction zones, pectin cell walls seemed to lose their integrity due to dissolution (Figure 5B,C). Reproductive structures, such as conidiophore, were found in the adaxial epidermis (Figure 5D). In the substomatic chamber, C. coffeicola hyphae were found as curling structures. The palisade parenchyma in the lesioned region seems to undergo cell wall lysis (Figure 5E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63432/63432fig01.jpg" /> 
Figure 1: Details of the individual steps in the protocol to harvest lesioned tissues. Harvest the piece of the lesion with a scalpel and a tweezer. Immerse the leaf samples into the fixative solution. Subject the samples to vacuum pumping and rotation. Follow the protocol for the dehydration and embedding processes. The polymerized sample is sectioned in a rotative microtome. The slides with lesion sections are mounted and stained to verify fungal hyphae and pectin-rich cell walls using the double-staining method. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63432/63432fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63432/63432fig02.jpg" /> 
Figure 2: Details of the individual steps of the sample section before double staining. (A) Fixation step. (B) Dehydration in graded ethanol series; the leaf tissue is incubated in ethanol for 15 min at each concentration. (C) Polymerization inside the plastic mold. (D) Block of polymerized sample glued to a piece of wood. (E) Wood-glued block positioned on the microtome for the sectioning process. (F) Tissue sections on the slide (denoted by arrows). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63432/63432fig02large.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/63432/63432fig03.jpg" /> 
Figure 3: Details of the individual steps of the double-staining protocol. (A) Cover the sections with drops of cotton blue lactophenol and heat the slides on a hot plate at 45 &#176;C. (B-C) Remove the excess dye by washing in distilled water. (D) Sections on the glass slides after washing (denoted by arrows). (E) Cover the sections with drops of ruthenium red. (F-G) Remove the excess dye by washing in distilled water. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63432/63432fig03large.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/63432/63432fig04.jpg" /> 
Figure 4: Histochemical double staining protocol for pectin and fungal structures in coffee rust lesion. (A-C) Sections stained only with cotton blue lactophenol. Fungal hyphae are stained in dark blue (arrows). The haustorium mother cell (Hmc) and haustorium (Ha) have a dark blue color. (D-H) Double staining using cotton blue lactophenol and ruthenium red. (D) Overview of pustule (Pu) on a leaf. (E-F) Haustorial neck (arrows) with pectin (pink-red color). (G) Arrows indicate the beginning of pectin-rich encapsulation of haustorium. (H) Complete encapsulation of haustorium by pectin (arrows). Epi Aba - Epidermis abaxial; Epi Ada - Epidermis adaxial; Sp - Spongy parenchyma; Pp - palisade parenchyma. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63432/63432fig04large.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/63432/63432fig05.jpg" /> 
Figure 5: Histochemical double staining protocol for pectin and fungal structures in cercosporiosis lesions in coffee mesophyll tissues. (A) Lesion border without fungi. (B-F) Cross-sections of the lesioned leaf. (A) The integrity of pectin-rich cell walls of the spongy parenchyma. (B-D,F) In the infected tissues, the Cercospora coffeicola hyphae were evident (arrows) and caused damage in pectic cell walls. It was possible to verify the conidiophore under the cuticle (CT) (D). Epi Aba - Epidermis abaxial; Epi Ada - Epidermis adaxial; Sp - Spongy parenchyma; Pp - Palisade Parenchyma; St - Stomata. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63432/63432fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Double-Staining Method to Detect Pectin in Plant-Fungus Interaction

This protocol describes a microscopical method to detect pectin in coffee-fungus interaction.

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an ...

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4.5K Views.  University of São Paulo. This protocol describes a microscopical method to detect pectin in coffee-fungus interaction.

Video: Double-Staining Method to Detect Pectin in Plant-Fungus Interaction

Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls are composed of cellulose and non-cellulosic polysaccharides together with proteins, phenolic compounds and water. 90% of the cell wall is composed of&#160;polysaccharides (e.g., pectins) and arabinogalactan proteins (AGPs). The fluorescent immunolocalization of specific glycan epitopes in plant histological sections remains a key tool to uncover remodeling of wall polysaccharide networks, structure and components.
Here, we report an optimized fluorescent immunolocalization procedure to detect glycan epitopes from AGPs and pectins in plant tissues. Paraformaldehyde/glutaraldehyde fixation was used along with LR-White embedding of the plant samples, allowing for a better preservation of the tissue structure and composition. Thin sections of the embedded samples obtained with an ultra-microtome were used for immunolocalization with specific antibodies. This technique offers great resolution, high specificity, and the chance to detect multiple glycan epitopes in the same sample. This technique allows subcellular localization of glycans and detects their level of accumulation in the cell wall. It also permits the determination of spatio-temporal patterns of AGP and pectin distribution during developmental processes. The use of this tool may ultimately guide research directions and link glycans to specific functions in plants. Furthermore, the information obtained can complement biochemical and gene expression studies.

This protocol describes in detail how the plant material for immunolocalization of Arabinogalactan proteins and pectins is fixed, embedded in a hydrophilic acrylic resin, sectioned and mounted on glass slides. We show cell wall related epitopes will be detected with specific antibodies.

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls ...

Developmental Biology

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

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Bioengineering

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...

Experimental Screening Protocols, Immunocytochemistry and Microscopy-based Imaging Techniques for Penium margaritaceum

The cell wall is the first component of signal reception/transduction for a plant cell during development and when responding to environmental abiotic and biotic stressors. The cell constantly monitors the integrity of its cell wall and modulates it in response to stress. Elucidating the specific structural and biochemical modulations occurring in the cell wall is a difficult task especially when employing multicellular plants and their organs/tissues. This is due to limits as to what can be resolved in an individual cell that is part of a complex multicellular network. The unicellular streptophyte alga, Penium margaritaceum, has recently been used in investigations of pectin dynamics, cell wall-based phenotypic plasticity and multiple aspects of algal cell biology. Its simple phenotype, distinct cell wall that has many components notably similar to land plant cell walls, and ease in immunocytochemical and experimental studies make it a powerful model organism in plant cell wall biology. The goal of this study is to provide the basic techniques for culturing, experimental manipulation and screening of applied stressors. Screening protocols for immunocytochemistry, confocal laser scanning microscopy imaging and scanning electron microscopy imaging&#160;of cell wall structure. Likewise, many of the described techniques may be modified for a wide array of other cell and molecular studies.

This report describes the fundamental methods used to culture and experimentally manipulate the unicellular streptophyte alga, Penium margaritaceum. It also provides fundamental protocols of microscopy-based imaging, including live cell labeling with monoclonal antibodies and other fluorescent probes and scanning electron microscopy.

The cell wall is the first component of signal reception/transduction for a plant cell during development and when responding to environmental abiotic and ...

Double-Staining Method: A Technique to Visualize Plant-Fungus Interactions Using Ruthenium Red and Lactophenol Cotton Blue

Source: Marques, J. P. R., and Nuevo, L. G. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/63432">Double-Staining Method to Detect Pectin in Plant-Fungus Interaction.</a> J. Vis. Exp. (2022).
This video demonstrates a double staining method using ruthenium red and lactophenol cotton blue to visualize pectins and chitins during plant-fungus interactions in a coffee rust plant. The procedure helps understand plant defense mechanisms during fungal infections.

Source: Marques, J. P. R., and Nuevo, L. G. Double-Staining Method to Detect Pectin in Plant-Fungus Interaction. J. Vis. Exp. (2022).
This video ...

JoVE Encyclopedia of Experiments

Biological Techniques

Staining Techniques

Cell staining

Immunolocalization of Arabinogalactan Proteins and Pectins in Plant Tissue

Source: Costa, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61034">Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues.</a> J. Vis. Exp. (2021).
This video demonstrates an immunostaining technique to localize arabinogalactan proteins (AGPs) and pectins in plant tissues. Fixed and resin-embedded plant tissue sections are stained with AGP- and pectin-specific primary and fluorescent secondary antibodies and a cellulose-binding fluorescent dye to reveal their tissue-specific colocalization in the cell wall.

1. Immunolocalization
NOTE: The fluorescent immunolocalization procedure relies on sequential use of two antibodies. The primary antibody is raised ...

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

A Method for Sample Preparation to Visualize Biofilm Forming Bacteria on Fungal Hyphae

Source: Guennoc, Cora Miquel, et al, <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54771">A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy.</a> J. Vis. Exp. (2017).
This video describes a method to grow and qualitatively analyze bacterial biofilms on fungal hyphae using microscopy. Bacterial biofilms frequently form on fungal surfaces and involve numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation.

1. Preparation of an In Vitro Biofilm of Bacteria on the Fungal Colony

	Centrifuge the bacterial culture at 5,000 x g for 3 min and suspend the pellet in ...

Double-Staining Method to Detect Pectin in Plant-Fungus Interaction

Summary

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Experimental Screening Protocols, Immunocytochemistry and Microscopy-based Imaging Techniques for Penium margaritaceum

Double-Staining Method: A Technique to Visualize Plant-Fungus Interactions Using Ruthenium Red and Lactophenol Cotton Blue

Immunolocalization of Arabinogalactan Proteins and Pectins in Plant Tissue

A Method for Sample Preparation to Visualize Biofilm Forming Bacteria on Fungal Hyphae