Name: double-staining method to detect pectin in plant-fungus interaction
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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>
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Botany and Plant Biology. 20, 5 (1863).</li><li>Mangin, L. <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+Peronospores.">Research on the Peronospores.</a> Bulletin of the Natural History Society of Autun. 8, 55-108 (1895).</li><li>Underwood, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plant+cell+wall:+a+dynamic+barrier+against+pathogen+invasion.">The plant cell wall: a dynamic barrier against pathogen invasion.</a> Frontiers in Plant Science. 3 (85), 1-6 (2012).</li><li>Hu&#776;ckelhoven, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall-associated+mechanisms+of+disease+resistance+and+susceptibility.">Cell wall-associated mechanisms of disease resistance and susceptibility.</a> Annual Review of Phytopathology. 45, 101-127 (2007).</li><li>Voigt, C. 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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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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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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.

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	<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...

Watch this Scientific Journal Video about Double-Staining Method to Detect Pectin in Plant-Fungus Interaction at JoVE.com

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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