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

Summary

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

Abstract

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 µm thick sections, which were deposited on a glass slide covered with water and subsequently heated at 40 °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.

Introduction

Cell wall defense mechanisms in plants are crucial to restrain fungal infection. Studies have reported changes in cell wall thickness and composition since the 19^th century^1,2. 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 attack^4,5,6,7.

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 methylated^8,9. 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 enzymes^10,11,12. 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 resistance^13,14,15,16, 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 encasement^6,7.

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 crops^18,19. 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 have been reported. To the authors' 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 crops^21,22. This fungus causes necrotic lesions in both leaves and berries. After penetration, C. coffeicola colonizes plant tissues through intracellular and intercellular pathways^23,24,25.

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.

Protocol

1. Preparation of the buffering solution and reagents

1. 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.
2. 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.
3. 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.

2. Plant samples and fungus inoculation

NOTE: For experiments on leaves affected by coffee rust, five 2-month-old seedlings of Coffee arabica cv. Catuaí were grown and kept in a greenhouse at the Center of Nuclear Energy in Agriculture (CENA) of the University of São Paulo, Piracicaba, São Paulo State, Brazil.

1. 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 ± 3 °C with a 12 h photoperiod created by LED lamps at the photon flux of 250 µmol photons s^-1 m^-2. Replace the Hoagland nutrient solution every week for 4 months.
2. Inoculate four expanded leaves from five plants on their abaxial surfaces with 1 x 10³ 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.
3. Harvest characteristic lesions caused by Cercospora coffeicola from Coffea arabica cv. Obatã plants located (coordinates: -22.906506126269942, -47.015075902025266) at the Biological Institute, Campinas, São 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.

3. Sample harvesting, fixation, and dehydration

1. 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 and Figure 2A). The fixation step can take place in a refrigerator for 48 h.
2. 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).
3. 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).

4. Historesin embedding procedure

1. Gradually transfer the samples to glycol methacrylate (GMA) in three steps, following the manufacturer'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.
   1. Immerse the samples in 1:2 Solution A: 100% ethanol for 3 h.
   2. Immerse the samples in 1:1 Solution A: 100% ethanol for 3 h.
   3. 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.

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).

1. 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).
2. 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.
3. 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.
4. 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.
5. 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).

6. Sectioning

1. Using a rotative microtome equipped with 8 cm steel blades (Figure 2E), cut the block into 5 µm 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 °C to dry and promote the adhesion of the sections to the glass slides.
2. After drying (Figure 2F), label the glass slides with the block reference name and the slide number.

7. Double staining process

1. 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 °C for 5 min (Figure 3A).
2. Remove the excess dye by washing the slide three times in a beaker filled with distilled water (Figure 3B-D).
3. Stain with 2 mL of 0.01% ruthenium red in water for 1 min (Figure 3E).
4. Remove the excess dye by washing the slide three times in a beaker filled with distilled water (Figure 3F,G).
5. 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.

Representative Results

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).

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

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

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

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

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

Discussion

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 pathosystem^14,29. Among the cell wall polymers associated with defense mechanisms, pectin plays an important role in the plant-pathogen system^10,11,12. 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 Plasmodiophora brassicae in Arabidopsis thaliana³⁴, Elsinoë 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 dye^36,38,39,40. 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 acids^42,43,44. 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 pectin^8,9,45. 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' 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.

Materials

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