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: Th...

<ol>
	<li>deBary, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+on+the+development+of+some+parasitic+fungi.">Research on the development of some parasitic fungi.</a> Annals of Natural Sciences. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+wall.">The cell wall.</a> Biochemistry and Molecular Biology of Plants., 2nd Edition. , 45 (2015).</li><li>Lionetti, V., Cervone, F., Bellincampi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methyl+esterification+of+pectin+plays+a+role+during+plant-pathogen+interactions+and+affects+plant+resistance+to+diseases.">Methyl esterification of pectin plays a role during plant-pathogen interactions and affects plant resistance to diseases.</a> Journal of Plant Physiology. 169 (16), 1623-1630 (2012).</li><li>Lionetti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pectoplate:+the+simultaneous+phenotyping+of+pectin+methylesterases,+pectinases,+and+oligogalacturonides+in+plants+during+biotic+stresses.">Pectoplate: the simultaneous phenotyping of pectin methylesterases, pectinases, and oligogalacturonides in plants during biotic stresses.</a> Frontiers in Plant Science. 6 (331), 1-8 (2015).</li><li>Lionetti, 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=Three+pectin+methylesterase+inhibitors+protect+cell+wall+integrity+for+Arabidopsis+immunity+to+Botrytis.">Three pectin methylesterase inhibitors protect cell wall integrity for Arabidopsis immunity to Botrytis.</a> Plant Physiology. 173 (3), 1844-1863 (2017).</li><li>Heath, M. 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R., 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=Sugarcane+smut:+shedding+light+on+the+development+of+the+whip-shaped+sorus.">Sugarcane smut: shedding light on the development of the whip-shaped sorus.</a> Annals of Botany. 119 (5), 815-827 (2017).</li><li>Delaye, L., Garc&#237;a-Guzm&#225;n, G., Heil, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endophytes+versus+biotrophic+and+necrotrophic+pathogens-are+fungal+lifestyles+evolutionarily+stable+traits.">Endophytes versus biotrophic and necrotrophic pathogens-are fungal lifestyles evolutionarily stable traits.</a> Fungal Diversity. 60 (1), 125-135 (2013).</li><li>Avelino, 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=The+coffee+rust+crises+in+Colombia+and+Central+America+(2008-2013):+impacts,+plausible+causes+and+proposed+solutions.">The coffee rust crises in Colombia and Central America (2008-2013): impacts, plausible causes and proposed solutions.</a> Food Security. 7, 303-321 (2015).</li><li>Zambolim, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+and+management+of+coffee+leaf+rust+in+Brazil.">Current status and management of coffee leaf rust in Brazil.</a> Tropical Plant Pathology. 41, 1-8 (2016).</li><li>Swiderska-Burek, U., 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=Phytopathogenic+Cercosporoidfungi-from+taxonomy+to+modern+biochemistry+and+molecular+biology.">Phytopathogenic Cercosporoidfungi-from taxonomy to modern biochemistry and molecular biology.</a> International Journal of Molecular Sciences. 21 (22), 8555 (2020).</li><li>Andrade, C. 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=Infection+process+and+defense+response+of+two+distinct+symptoms+of+Cercospora+leaf+spot+in+coffee+leaves.">Infection process and defense response of two distinct symptoms of Cercospora leaf spot in coffee leaves.</a> Phytoparasitica. 49 (7), 727-737 (2021).</li><li>Zambolim, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coffee+tree+diseases.">Coffee tree diseases.</a> Handbook of Phytopathology: Diseases of cultivated plants. 5th ed. , 810 (2016).</li><li>Casta&#241;o, A. J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coffee+rust.">Coffee rust.</a> Informative report Cenicaf&#233;. 82, 313-327 (1956).</li><li>Echandi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coffee+rust,+caused+by+the+fungus+Cercospora+coffeicola.">Coffee rust, caused by the fungus Cercospora coffeicola.</a> Turrialba. 9 (2), 54-67 (1959).</li><li>Souza, A. G. C., Rodrigues, F. A., Maffia, L. A., Mizubuti, E. S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+process+of+Cercospora+coffeicola+on+coffee+leaf.">Infection process of Cercospora coffeicola on coffee leaf.</a> Journal of Phytopathology. 159 (1), 6-11 (2011).</li><li>Karnovsky, M. 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F., Amorim, L., Appezzato-da-Gl&#243;ria, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathological+evidence+of+concomitant+sexual+and+asexual+reproduction+of+Elsino&#235;+ampelina+in+grapevine+under+subtropical+climate.">Histopathological evidence of concomitant sexual and asexual reproduction of Elsino&#235; ampelina in grapevine under subtropical climate.</a> Physiological and Molecular Plant Pathology. 111, 101517 (2020).</li><li>Marques, J. P. R., Soares, M. K. M., Piracicaba, F. E. A. L. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Techniques Applied to Plant Histopathology. , 140 (2021).</li><li>Navarro, B. L., Marques, J. P. R., Appezzato-da-Gl&#243;ria, B., Sp&#243;sito, M. 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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 ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#237;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</stro...

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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 ...

This protocol is significant as it make it possible to identify the plant-fungus interaction. In addition to coloring the pectin, it also demonstrates defects on the pathogen cell wall polymer and allows the identification of fungus structures. This simple and uncomplicated technique can be performed using light microscopy and it doesn't require any expensive equipment such as scanning electron microscope.In addition, it is a fast technique that obtains an excellent distinction of plant structures for histopathological studies. The technique identifies the plant-fungus interaction. Hence, it collaborates to diagnose the diseases caused by biotrophic and necrotrophic fungi such as coffee rust and cercosporiosis.To begin, harvest and approximately 10 square millimeter leaf sample at the middle region of the lesion using a scalpel and tweezer, and immerse it in 30 milliliters of Karnovsky fixative solution. Subject the leaf sample to a low vacuum of 500 to 600 millibar for 15 minutes using an oil pump, at least four times to increase the permeability of the fixative solution in the leaf tissue. After fixation, wash the leaf sample three times in 0.5 M cacodylate buffer diluted in distilled water for five minutes each, and then transfer it to a graded ethanolic series for 15 minutes at each ethanol concentration.To gradually transfer the samples to glycol methacrylate, make Solution A by mixing the one gram of glycol methacrylate powder with 100 milliliters of basic resin under magnetic agitation. Then, immerse the samples in ratio one to two of Solution A and 100%ethanol for three hours. Immerse the samples in ratio one to one for Solution A:100%ethanol for three hours, then immersed in a pure basic resin for two to four days.Subject the sample to a low vacuum at least four times a day for 15 minutes, followed by rotation. Mix 15 milliliters of Solution A with one milliliter of the hardener in a beaker with rotation for two minutes to produce the polymerization Solution B.Put 1.2 milliliters of this polymerization Solution B in plastic molds. Using a wooden pick, transfer the lesioned leaf samples from pure basic resin to Solution B and avoid using tweezers as they can cause tissue crushing.Ensure to quickly orient leaf samples perpendicular to the plastic molds as Solution B quickly becomes viscous. When the perpendicular orientation of the leaf samples is achieved, wait for 30 minutes and then transfer the plastic mold to a plastic or glass chamber containing silica gel to prevent humidity. Once the resin and the leaf sample are polymerized after two to three hours, 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. Cut the block into five micrometer thick sections using a rotating microtome equipped with eight centimeter steel blades. Place the sections onto glass slides covered with distilled water.Then, transfer the slides to a hot plate to dry at 40 degrees Celsius and promote the adhesion of the sections to the glass slides. After drying, label the glass slides with the block reference name and the slide number. Cover the sections with two milliliters of 5%cotton blue in lactophenol and heat them on a hot plate at 45 degrees Celsius for five minutes.Remove the excess dye by washing the slide three times in a beaker filled with distilled water. Stain it with two milliliters of 0.01%ruthenium red in water for one minute. Remove the excess dye by washing the slide three times in a beaker filled with distilled water.Put a drop of distilled water over the sections and cover the sections with a cover slip for performing light microscopy analysis. In the double staining method, Hemileia vastatrix hyphae containing cell walls and the dense protoplasts content appeared dark blue in both spongy and palisade parenchyma. The haustorium mother cell and the haustoria also exhibited a dark blue color.The counter staining with ruthenium red clearly defined fungal distribution in the intercellular spaces. During the interaction, Hemileia vastatrix haustorium mother cell broke the host cell wall and developed a haustorial neck surrounded by pectic compounds. The pectin-rich pink-red colored encapsulation of the haustorial neck cannot prevent haustorial formation.In some cases, the pectin-rich encapsulation encased the haustorium incompletely. And in some cases, the haustorium is fully encapsulated. The pectin-rich cell wall maintain the integrity at the lesion border, where the fungus is not present.The double staining method demonstrated the presence of intercellular hyphae. In interaction zones, pectin cell walls appeared to lose their integrity due to dissolution. Reproductive structures such as conidia were found in the adaxial epidermis.Cercospora coffeicola hyphae were found in the substomatic chamber as curling structures, and the palisade parenchyma in the lesion region seems to undergo cell wall lysis. Avoid using tweezers as they can cause tissue crushing. 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.

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Research

JoVE Journal

Biology

4.1K Görüntüleme.  University of São Paulo. Bu protokol, bitki-mantar etkileşimini tanımlamayı mümkün kıldığı için önemlidir. Pektinin renklendirilmesine ek olarak, patojen hücre duvarı polimerindeki kusurları da gösterir ve mantar yapılarının tanımlanmasına izin verir. Bu basit ve karmaşık olmayan teknik, ışık mikroskobu kullanılarak gerçekleştirilebilir ve taramalı elektron mikroskobu gibi pahalı ekipmanlar gerektirmez.Ek olarak, histopatolojik çalışmalar için bitki yapılarının mükemmel bi...