Plant Cell Wall Changes in Common Wheat Roots as a Result of Their Interaction with Beneficial Fungi of Trichoderma

Plant cell walls play an important role in shaping the defense strategies of plants. This research demonstrates the influence of two differentiators: the lifestyle and properties of the Trichoderma species on cell wall changes in common wheat seedlings. The methodologies used in this investigation include microscopy observations and immunodetection. In this study was shown that the plant cell wall was altered due to its interaction with Trichoderma. The accumulation of lignins and reorganization of pectin were observed. The immunocytochemistry indicated that low methyl-esterified pectins appeared in intercellular spaces. Moreover, it was found that the arabinogalactan protein epitope JIM14 can play a role in the interaction of wheat roots with both the tested Trichoderma strains. Nevertheless, we postulate that modifications, such as the appearance of lignins, rearrangement of low methyl-esterified pectins, and arabinogalactan proteins due to the interaction with Trichoderma show that tested strains can be potentially used in wheat seedlings protection to pathogens.


Introduction
Common wheat (Triticum aestivum L.) is one of the most widely cultivated crops in the world. Statistics indicate that in 2018-2019, global wheat production was 762.4 million tons [1]. However, wheat production is mainly affected by the incidence of foliar and roots disease caused by pathogens, such as fungi from different genera, including Fusarium, Puccinia, Bipolaris sorokiniana, Zymoseptoria tritici, and others [2]. From an environmental perspective, it would be beneficial to use biological control agents (BCAs), serving as natural antagonists to phytopathogens [3]. The most studied and promising microorganisms used in a biocontrol system are the Trichoderma species [4,5]. Trichoderma strains used as biocontrol agents created a positive impact on plants, nutrient uptake, fertilizer use efficiency, growth, and rate of seed germination. These fungi triggered induced systemic resistance (ISR) in monocots [6,7] and dicots [8][9][10]. Data showed that these fungi can also stimulate a systemic acquired resistance (SAR) in plants [11]. Fungi of the genus Trichoderma (teleomorph Hypocrea) colonize different ecological niches and are free-living organisms. Habitats occupied by these fungi include rotting wood, soil, and the rhizosphere [4,12,13]. Trichoderma spp. influence pathogens, such as oomycetes (Oomycota), bacteria, viruses, and pathogenic fungi via hyperparasitism, competition, and antibiosis [14,15]. Our recent studies have revealed that Trichoderma spp. can suppress mycotoxin production by Fusarium species on natural substrates, exhibit mycoparasitic behavior, such as coiling Pure cultures of T. atroviride AN35 and T. cremeum AN392 were recultured from stock slants to 9 cm diameter Petri dishes with potato dextrose agar (PDA medium, Oxoid™ Thermo Fisher Scientific, Waltham, MA, USA). The fungal material was incubated at 23 • C in 12 h/12 h darkness/light conditions. The inoculum constituted only spores (without PDA disc) from an area of 5 mm of diameter from the 7-day old culture. Each plant treated with Trichoderma was inoculated with spores from 1 area. Control and inoculated with fungi plants were grown for 14 days at a temperature of 23 • C under 12 h/12 h darkness/light conditions in a growth chamber. After this time, the material was sampled. All of these steps were carried out under sterile conditions.

Microscopy Observations
Each variant for microscopy observations were replicated from 7 to 10 plants. Roots from this same plant root system were divided-one (root hairs zone) for light and confocal microscopy. Three sections were taken for each staining. Second root (root hairs zone) were taken for scanning electron microscopy (SEM). Figure 1 shows a scheme of the experiment.

Preparation of Plant Material for Electron Scanning Microscopy
After 14 days of incubation with Trichoderma spp., the roots of wheat seedlings were fixed in a mixture of 4% methanol-free formaldehyde (Polysciences, Hirschberg an der Bergstrasse, Germany), 0.5% glutaraldehyde (Polysciences, Hirschberg an der Bergstrasse, Germany), and phosphate-buffered saline (PBS) at 4 • C for 24 h. SEM measurements were realized using the Quanta FEG 250 (FEI) microscope under low vacuum mode at the pressure of 70 Pa. Low vacuum conditions were realized by the dosing of ultra-pure water vapor. This prevents the samples from charging, thus, no coating was used. The plant material was frozen in liquid nitrogen and lyophilized for 4 h. SEM imaging was realized using 10 kV beam acceleration voltage with the 30 µm aperture, at a typical working distance of 10 mm.

Preparation of Plant Material for Light and Confocal Microscopy Observations
After 14 days of inoculations, the roots of the wheat seedlings were fixed as mentioned above. Then, the material was first dehydrated in an ethanol series (10%, 30%, 50%, 70%, 80%, 90%, 96%, 100%), then dehydrated in a solution containing 100% acetone and embedded in Technovit 8100 resin (Kulzer GmbH, Wehrheim, Germany) in line with the manufacturer's instructions. The samples were sectioned on a rotary microtome (Leica, RM2125RTS) on 5 µm thin sections. Transverse root sections were collected on microscope slides coated with poly-l-lysine (Merck KGaA, Darmstadt, Germany, formerly Sigma, St. Louis, MO, USA).

Preparation of Plant Material for Electron Scanning Microscopy
After 14 days of incubation with Trichoderma spp., the roots of wheat seedlings were fixed in a mixture of 4% methanol-free formaldehyde (Polysciences, Hirschberg an der Bergstrasse, Germany), 0.5% glutaraldehyde (Polysciences, Hirschberg an der Bergstrasse, Germany), and phosphatebuffered saline (PBS) at 4 °C for 24 h. SEM measurements were realized using the Quanta FEG 250 (FEI) microscope under low vacuum mode at the pressure of 70 Pa. Low vacuum conditions were realized by the dosing of ultra-pure water vapor. This prevents the samples from charging, thus, no coating was used. The plant material was frozen in liquid nitrogen and lyophilized for 4 h. SEM imaging was realized using 10 kV beam acceleration voltage with the 30 µm aperture, at a typical working distance of 10 mm.

Preparation of Plant Material for Light and Confocal Microscopy Observations
After 14 days of inoculations, the roots of the wheat seedlings were fixed as mentioned above. Then, the material was first dehydrated in an ethanol series (10%, 30%, 50%, 70%, 80%, 90%, 96%, 100%), then dehydrated in a solution containing 100% acetone and embedded in Technovit 8100 resin (Kulzer GmbH, Wehrheim, Germany) in line with the manufacturer's instructions. The samples were sectioned on a rotary microtome (Leica, RM2125RTS) on 5 µm thin sections. Transverse root sections were collected on microscope slides coated with poly-L-lysine (Merck KGaA, Darmstadt, Germany, formerly Sigma, St. Louis, MO, USA).

Detection of Lignins, Pectins, and Colonization of Wheat Roots with Trichoderma Hyphae
The staining procedure of wheat roots treated with Trichoderma was carried out according to Chacón et al. [41]. The sections of roots were treated with 0.5% toluidine blue solution (Kolchem, Lodz, Poland) dye for 3 min, for detecting lignin, pectin, and hyphae of Trichoderma inside the wheat roots. The observations were captured using a light microscope (Olympus CX-41-1 with UC-30 camera, Olympus, Japan). Pectins were dyed a red-purple color, lignins a blue color.

Immunolabeling of Pectins, Hemicellulose, and Arabinogalactan Proteins
The detection process was carried out in sections using primary monoclonal antibodies-LM19 epitope (PlantProbes, Leeds, UK, Table 1) for low methyl-esterified pectins, and LM27 epitope (PlantProbes, Leeds, UK, Table 1) for grass xylan hemicelluloses. The arabinogalactan proteins (AGPs) were detected by monoclonal antibodies-JIM13, JIM14, LM2 (PlantProbes, Leeds, UK, Table 1). The immunolabeling procedure was carried out according to Lenartowska et al. [42] with modifications. The sections on the glass slides were first blocked in 2% bovine serum albumin (BSA, Merck KGaA, Darmstadt, Germany, formerly Sigma, St. Louis, MO, USA) in PBS for 3 h and then incubated with primary monoclonal antibodies overnight, diluted 1:20, washed 3 times with PBS buffer and incubated with goat anti-Rat IgG (H+L) cross-adsorbed secondary antibody-diluted in 1:100 conjugated with Cy3 (A10522, Thermo Fisher Scientific, Waltham, MA, USA, excitation: 550 nm, emission: 570 nm) for 1.5 h. In the end, the sections were washed 3 times with PBS buffer and 4 times with deionized water. The samples were coated with Citifluor™ AF1 (Electron Microscopy Science, Hatfield, PA, USA), closed with cover glass, and protected by nail polish. Observations were carried out using a confocal microscope (Olympus FV1200, Tokyo, Japan).
JIM14 unknown structure of epitope [43] LM2 carbohydrate epitope containing b-linked glucuronic acid [43] 2.2.5. Masking of Xylans by Pectins The detection process of masking xylans epitope LM27 by pectins was carried out according to Davies et al. [44] with modifications. The sections on the glass slides were first pre-treated with 2% pectinase (Merck KGaA, Darmstadt, Germany, formerly Sigma, St. Louis, MO, USA) in PBS buffer for 30 min, washed 3 times with PBS buffer. Then, the detection process of xylans with use of LM27 antibody, was carried out as described above. Some sections were carried out with control reaction using LM19 antibody.

Colonization of Wheat Roots by Trichoderma spp.
Transverse section of wheat root showed tissues: rhizodermis and root hairs, parenchyma cells, endodermis, pericycle and vascular bundle: xylem and phloem ( Figure 2). Scanning electron microscopy and the light microscopy (where sections were dyed by toluidine blue) observations indicated that both T. atroviride AN35 and T. cremeum-AN392 can colonize the roots of both cultivars of wheat seedlings. Fungal hyphae growth on the surface of roots ( Figures 3C and 4C for T. atroviride AN35-green arrows and Figures 3E and 4E for T. cremeum AN392-orange arrows). Fungal hyphae was also visible inside the root parenchyma cells ( Figures 3D and 4D for T. atroviride AN35 and Figures 3F and 4F for T. cremeum AN392). The light microscopy observations revealed that hyphae growth in the lumen of parenchyma ( Figures 5D and 6C,D for T. atroviride AN35, Figures 5F and 6F for T. cremeum AN392) and rhizodermis cells ( Figure 5D) was seen. The hyphae were in the apoplast and grew from cell-to-cell, with barriers such as the plant cell walls ( Figure 6C,F). for T. cremeum AN392) and rhizodermis cells ( Figure 5D) was seen. The hyphae were in the apoplast and grew from cell-to-cell, with barriers such as the plant cell walls ( Figure 6C,F).
Observations of fungal hyphae were based on morphological differences between control and material treated with Trichoderma. In SEM, hyphae are visible, like thin, long, and divided structures, which are not present in the control root. On micrographs taken on light microscopy, sections of fungal hyphae are visible, oval or long curved structures, which are not present in the control.

Pectins and Lignins
The shapes of cells in the control group were regular. Nevertheless, after interaction with both the fungi, the shapes of the cells became more irregular. Moreover, the deposition of the materials in the plant cell walls was different in the experimental group as compared to the deposition in the control group. The main component of the plant cell walls of wheat seedlings in the control group

Immunolocalization of Pectins
Immunolabeling via LM19 antibodies indicated that unesterified homogalacturonan was deposited in the cell walls of the control and experimental. It was also observed that the pectin epitope-LM19 was less abundant in the rhizodermis. In the material treated with Trichoderma spp., the accumulation of pectins was visible in the intercellular spaces, and this deposition contained unesterified homogalacturonan (Figures 7 and 8). In cultivar Bombona after interaction with T. cremeum AN392, a cell wall thickening labeled by LM19 antibody was observed ( Figure 7E). Observations of fungal hyphae were based on morphological differences between control and material treated with Trichoderma. In SEM, hyphae are visible, like thin, long, and divided structures, which are not present in the control root. On micrographs taken on light microscopy, sections of fungal hyphae are visible, oval or long curved structures, which are not present in the control.

Pectins and Lignins
The shapes of cells in the control group were regular. Nevertheless, after interaction with both the fungi, the shapes of the cells became more irregular. Moreover, the deposition of the materials in the plant cell walls was different in the experimental group as compared to the deposition in the control group. The main component of the plant cell walls of wheat seedlings in the control group and experimental group were pectins. However, after interactions with Trichoderma spp., pectins appeared in intercellular spaces (red-purple color and red arrows, Figures 5D and 6E). In the parenchyma cells, lignins were visible (blue color and blue arrows, Figures 5 and 6). It was also observed that the cell walls were thickened by pectins (Figures 5C and 6D) and lignins ( Figure 6D).

Immunolocalization of Pectins
Immunolabeling via LM19 antibodies indicated that unesterified homogalacturonan was deposited in the cell walls of the control and experimental. It was also observed that the pectin epitope-LM19 was less abundant in the rhizodermis. In the material treated with Trichoderma spp., the accumulation of pectins was visible in the intercellular spaces, and this deposition contained unesterified homogalacturonan (Figures 7 and 8). In cultivar Bombona after interaction with T. cremeum AN392, a cell wall thickening labeled by LM19 antibody was observed ( Figure 7E).

Immunolocalization of Hemicelluloses
Immunolabeling method using the LM27 antibody, specifically for grass xylans, showed that this fraction of hemicellulose in the roots of wheat seedling was mainly present in the root hairs, rhizodermis, and parenchyma cells (Figures 9 and 10). Analysis of masking xylans LM27 epitope by pectins showed that after using of pectinase pectins LM19 epitope were removed from this samples ( Figures 11A,B and 12A,B). Nevertheless, analysis showed that xylans LM27 epitope was masked by pectins LM19 epitope in tissues: endodermis, pericycle, vascular cylinder, phloem, and xylem; however, in this tissue, xylans were less abundant (Figures 11 and 12). Nevertheless, even after the interaction with both the Trichoderma spp., this xylan fraction remained unchanged.

Immunolocalization of Hemicelluloses
Immunolabeling method using the LM27 antibody, specifically for grass xylans, showed that this fraction of hemicellulose in the roots of wheat seedling was mainly present in the root hairs, rhizodermis, and parenchyma cells (Figures 9 and 10). Analysis of masking xylans LM27 epitope by pectins showed that after using of pectinase pectins LM19 epitope were removed from this samples ( Figure 11A,B and Figure 12A,B). Nevertheless, analysis showed that xylans LM27 epitope was masked by pectins LM19 epitope in tissues: endodermis, pericycle, vascular cylinder, phloem, and xylem; however, in this tissue, xylans were less abundant (Figures 11 and 12). Nevertheless, even after the interaction with both the Trichoderma spp., this xylan fraction remained unchanged.

Immunolocalization of Arabinogalactan Proteins
The AGPs were detected by monoclonal antibodies-JIM13, JIM14, and LM2. The results demonstrated that in the wheat seedlings, the AGP epitope-JIM13 occurred in the cell walls, in the

Immunolocalization of Arabinogalactan Proteins
The AGPs were detected by monoclonal antibodies-JIM13, JIM14, and LM2. The results demonstrated that in the wheat seedlings, the AGP epitope-JIM13 occurred in the cell walls, in the root hair, and each tissue in the control conditions and after interactions with T. atroviride AN35 and T. cremeum AN392. The AGPs epitope, JIM13, was present inside of the parenchyma and rhizodermis cells in all variants (Figures 13 and 14). The AGP epitope, LM2, was present in trichoblasts and root hair in parenchyma cells without the first layer under the rhizodermis and stele tissues in the control and experimental groups (Figures 15 and 16). The AGP epitope, JIM14, in the control condition was mostly visible in xylem. However, after interaction with both the Trichoderma spp., localization of this epitope was modified. It was strongly visible in parenchyma cells, especially in the cell walls of a few layers of parenchyma cells under the rhizodermis. The AGPs of this epitope were also detected in the cell walls of the rhizodermis and could be accumulated in the cytoplasm of the rhizodermis cells (Figures 17 and 18).  (Figures 13 and 14). The AGP epitope, LM2, was present in trichoblasts and root hair in parenchyma cells without the first layer under the rhizodermis and stele tissues in the control and experimental groups (Figures 15 and 16). The AGP epitope, JIM14, in the control condition was mostly visible in xylem. However, after interaction with both the Trichoderma spp., localization of this epitope was modified. It was strongly visible in parenchyma cells, especially in the cell walls of a few layers of parenchyma cells under the rhizodermis. The AGPs of this epitope were also detected in the cell walls of the rhizodermis and could be accumulated in the cytoplasm of the rhizodermis cells (Figures 17 and 18).

Discussion
The results demonstrated that both T. atroviride AN35 and T. cremeum AN392 colonize the roots of wheat seedlings. The microscopy analysis showed that the hyphae of both the Trichoderma grew on the outer layer of the roots of the wheat seedlings. This behavior was also observed in T. atroviride during the colonization of Arabidopsis roots [45]. The present study has also showed that T. atroviride AN35 and T. cremeum AN392 grew in the apoplast, in intercellular spaces, and into the parenchyma cells of wheat roots. The same behavior was observed for other Trichoderma strains in their interaction with different plants. For example, it was shown that, Trichoderma harzianum CECT 2413 grew into the roots of tomato [41], and also colonized olive roots [46].
This study revealed that the composition of the plant cell walls of wheat seedling roots changed after their interaction with T. atroviride AN35 and T. cremeum AN392. Yedida et al. [47] showed that electronically dense material was found in intercellular spaces of cucumber roots after their interaction with Trichoderma. Results obtained in this study have shown that pectins reorganized themselves after interacting with T. atroviride AN35 and T. cremeum AN392. Pectins appeared in

Discussion
The results demonstrated that both T. atroviride AN35 and T. cremeum AN392 colonize the roots of wheat seedlings. The microscopy analysis showed that the hyphae of both the Trichoderma grew on the outer layer of the roots of the wheat seedlings. This behavior was also observed in T. atroviride during the colonization of Arabidopsis roots [45]. The present study has also showed that T. atroviride AN35 and T. cremeum AN392 grew in the apoplast, in intercellular spaces, and into the parenchyma cells of wheat roots. The same behavior was observed for other Trichoderma strains in their interaction with different plants. For example, it was shown that, Trichoderma harzianum CECT 2413 grew into the roots of tomato [41], and also colonized olive roots [46].
This study revealed that the composition of the plant cell walls of wheat seedling roots changed after their interaction with T. atroviride AN35 and T. cremeum AN392. Yedida et al. [47] showed that electronically dense material was found in intercellular spaces of cucumber roots after their interaction with Trichoderma. Results obtained in this study have shown that pectins reorganized themselves after interacting with T. atroviride AN35 and T. cremeum AN392. Pectins appeared in intercellular spaces, and the immunocytochemistry reaction showed that it could be an unesterified homogalacturonan.
Deesterified pectins (JIM5 epitope) were detected in the cell wall of Allium porrum in interaction with the arbuscular mycorrhizal fungus (AMF), Glomus versiforme [48], and in Corylus avellana, treated with Tuber magnatum [49]. It has been documented that the esterification of pectins also occurs while plants interact with pathogenic fungi. The lower activity level of pectin methyl esterases (PME), response for modulation, and the degree and patterns of methyl esterification of pectins caused a higher degree of methyl esterification of pectins in the cell walls of transgenic durum wheat (with a higher expression of pectin methyl esterase inhibitor (PMEI)-antagonist of pectin methyl esterases (PME), thereby increasing the resistance of these plants to pathogens like B. sorokiniana or Fusarium graminearum. A higher degree of methyl esterification caused more resistance to fungal endopolygalacturonases (PGs) [50]. It was also shown that in resistant wheat plants, the activity of the WheatPME1 gene was downregulated in the resistance line to Fusarium Head Blight (FHB) and induced in the susceptibility line. During the early stages of Fusarium infection in wheat plants, the level of methyl esterification was lower [51]. Moreover, pectin deesterification can be involved in a plant's defense strategy, in the first steps of pathogen infection [52] as pectins are a source of oligogalacturonides (OG)-important endogenous molecules, which are known to play a crucial role in the plant's defense response to pathogens. Oligogalacturonides are short fragments of homogalacturonan caused by microbial and endogenous polygalacturonidases [53]. The pattern of methyl esterification is critical for the action of these enzymes. Oligogalacturonides need to be deesterified to elicit a defense response [54]. In strawberry plants, oligogalacturonides with a low degree of methyl esterification were correlated with the defense response to Botrytis cinerea [54]. Nevertheless, it was shown that acetylation of OG reduced the haustoria of Blumeria graminis wheat leaves [55]. Taking into account that the currently studied strains Trichoderma-mycoparasitic T. atroviride and saprotrophic T. cremeum are not pathogens of plants, it can be assumed that reorganization of pectins included low methyl-esterified pectins in cell walls of wheat roots after interaction with Trichoderma strains is a manifestation of symbiotic interactions.
The results obtained in this research showed that in the parenchyma cells of wheat roots, lignins are formed as a result of the interaction with both the Trichoderma. Deposition of these molecules in the plant cell walls makes them more resistant to cell wall degrading enzymes (CWDEs), spread the toxins into the host [32], and increases their resistance to pathogens, like the increased resistance of cotton plants to Verticillium dahliae [56] and Eucalyptus plants to Mycosphaerella fungi [57]. The data indicate that following the interaction of plants with pathogens, the genes responsible for lignin biosynthesis are upregulated [58,59]. Chacón et al. [41] demonstrated that the colonization of tomato roots by T. harzianum leads to modification in the cell walls in the form of lignin depositions. Lignins were also present in cucumber shoots after their interaction with T. atroviride TR25 [60]. We suppose that lignin deposition in wheat roots after treated with tested Trichoderma strains showed positive impact on this plants. After interaction with T. atroviride AN35 and T. cremeum AN392 deposited lignins in wheat plant cell wall constitutes a physical barrier to pathogen invasion into wheat roots. It was also shown that inoculation of cucumber roots by T. atroviride TR25 can trigger on callose deposition in shoots of this plant [60]. The data indicates that callose was synthesized in plant cell walls after pathogen infection, it is believed that depositions of callose is physically protect plants by pathogen invasion [61]. On the other side, Nishimura et al. [62] showed that in Arabidopsis mutant: powdery mildew resistant 4 (pmr4), lacking pathogen-induced callose, despite this, is resistant to pathogens. They showed that in plants mutants after infection by pathogen, genes connected with on salicylic acid (SA) pathway were up-regulated. They conclude that callose synthesis affected negatively on salicylic acid (SA) pathway [62], which take a role in systemic acquired resistance (SAR) [63]. They also showed that in mutant plants papilla like structures were appear in plant cell walls after pathogen infection, but they are lacking in callose deposition [62].
The microscopy observations in this study revealed that in the control wheat roots and the wheat roots exposed to both Trichoderma hemicelluloses, the xylans epitope-LM27 in roots are present in the rhizodermis and parenchyma cells. However, data show that hemicelluloses can be masked by pectins [64]. Microscopy observations in this study showed that in wheat seedlings xylans LM27 epitope can be masked by pectins LM19 epitope in tissues like: endodermis, pericycle, vascular cylinder, phloem, and xylem. Nevertheless, in these tissues, xylans are less abundant. Hemicelluloses can play a role in the plant's defense strategy to pathogens. Data show that the expression of gene encoding enzymes responsible for xylan biosynthesis makes plants more resistant to infection by B. graminis [31]. Changes in acetylation of xylans and pectins also influence a plant's resistance to pathogens. Transformation of Arabidopsis and Brachypodium distachyon to express acetylesterases from Aspergillus nidulans increased the resistance of these plants to the fungal pathogens Botrytis cinerea and Bipolaris sorokiniana [65]. Data indicate that transgenic durum wheat produces xylanase inhibitors to counteract microbial xylanases. Expression of these enzymes in plants increased their resistance to F. graminearum, but do not to B. sorokiniana [66]. However, the results obtained in this research did not reveal changes in the reorganization of the xylan LM27 epitope after interaction with Trichoderma. With this in mind, it is believed that xylans epitope LM27 did not take a role in plant cell wall interaction with beneficial fungi of Trichoderma, but this does not exclude the involvement of other hemicelluloses in these interactions, which, however, would have to be confirmed in further analyzes.
AGPs play diverse roles in a plant's development, growth, defense strategy, and also in the colonization of plant roots by different microorganisms [37]. Each of the recognized epitope of the AGPs by the antibody is located in a specific tissue on plant organ [35]. The results obtained in this research showed that, in wheat seedlings, after their interaction with Trichoderma, the AGP epitope JIM13 occurred in all types of root tissues in the control group and experimental group. Nevertheless, [67] showed that after the interaction of Alnus spp. with nitrogen-fixing symbiotic Frankia, the AGP epitope, JIM13, appeared in a lot of in the symbiotic interface at the membrane cell wall of the border cells. The AGP JIM13 epitope was also detected in the cytoplasm and vacuole at a mature stage of infection. The subcellular localization of JIM13 suggests that these AGPs can play a role in the proliferation of Frankia in the nodule. Berry et al. [67] also demonstrated that AGPs can interact with partially methyl-esterified epitopes of homogalacturonan, epitope JIM7, which were found in colocalization with the AGP epitope JIM4. The result obtained in our study indicated that in wheat roots, the AGP epitope, JIM14, can play a role in response to Trichoderma. In roots treated with fungi, proteins of this epitope abundantly occurred in the cell walls of parenchyma and of rhizodermis and cytoplasm of rhizodermis. The data showed that other AGPs can play a role in the interaction of plants with pathogens or symbiotic microorganisms. A study that focused on the interaction between mycorrhizal AM Glomus fungi and Medicago truncatula showed that in cells with mycorrhiza abundant transcripts of AGPs were found [68]. However, it was found that Arabidopsis with a mutation in the AGP17 gene was not transformed by Agrobacterium tumefaciens. Treated wild plants with Yariv reagent also demonstrated a reduced frequency of transformation by Agrobacterium. Nevertheless, recovering the AGP 17 gene from wild plants restored the mutant's ability to transform [69]. Moreover, the microscopic observations obtained in this research revealed that localization of the tested AGPs, such as JIM13 (in all tissues), LM2 (in trichoblasts and root hairs, and parenchyma cells without the first layer under the rhizodermis), and JIM14 (mostly in xylem) in the control group showed localization of AGP epitopes similar to that in barley roots [35], and can play a role in wheat root development. Thus, the data indicate that AGPs can act as a part of plant response to both symbiotic and pathogenic microorganisms. Here, it can be assumed that wheat AGP epitope JIM14 was involved in establishing of symbiotic interactions with fungi of T. atroviride AN35 a and T. cremeum AN392.
The obtained results showed that both the tested Trichoderma species can colonize the roots of wheat seedlings. This interaction triggered changes in plant cell walls in the form of lignin depositions and rearrangement of pectins, including low methyl-esterified pectins. This research also showed that the AGP epitope JIM14 can participate in the wheat seedling-Trichoderma interactions. Regarding previous reports, it is concluded that the changes in plant cell walls observed here in wheat seedlings as a result of interaction with T. atroviride AN35 and T. cremeum AN392 and their potential to colonize the root endosphere of wheat may indicate ability of these strains to form symbiotic interactions with wheat. Bearing in mind the non-pathogenicity of these fungi towards plants, which has been numerously documented previously [3,4], it can be assumed that the tested strains may have a beneficial effect on wheat plants. Moreover, these and their previously documented antagonistic abilities against Fusarium species, including the potential to produce antifungal volatile metabolites and lytic enzymes [16,23,70,71], may predispose these Trichoderma strains to be used as potential biological control agents. Nevertheless, further experiments with the use this two Trichoderma strains in field conditions, extended with the analysis of the impact of both fungi on the wheat hormonal metabolism, as suggested by research of Nishimura et al. [63], are required to approved the beneficial effects of T. atroviride AN35 and T. cremeum AN392 on wheat plants and their potential for use in biological control.