Modifications of the CW during the formation of the SFS induced by Sedentary Endoparasitic Nematodes

Although in early stages the cells that give way to the formation of giant cells and the syncytia undergo similar changes, throughout the period of formation of SEAs the progressive molecular dialog between the nematode and its host presents distinctive features, depending on whether it is a nematode that induces the formation of giant cells or syncytia; this in turn produces structural differences between both types of SFS. Changes in the CW include modifications in the thickness and extension of the CW, as well as the formation of invaginations in areas adjacent to the xylem vessesl, both in giant cells and in syncytia.

In the formation of giant cells, the initial expansion zones where the thickening of the CW begins, are the first to form; these changes then extend to cover large areas of it (^{Rodiuc et al., 2014}). The mechanisms used to regulate the deposition of new material to the CW and its thickening vary, and as the giant cells form, highly reticulated regions are created from these expansion zones that resemble CW labyrinths called "invaginations", through which the transportation of solutes into and out of the giant cell intensifies (^{Vieira et al., 2012}; ^{Rodiuc et al., 2014}). Such invaginations develop throughout the process of maturation of the nematode and degenerate once it reaches its maturity and completes its life cycle (^{Rodiuc et al., 2014}).

There are reports that reveal the importance of the plasmodesmata during the formation of the SFS. Plasmodesmata are membrane canals located in the cell walls, which provide cytoplasmic continuity between cells, creating a network of intercellular exchange. In a natural way, these plasmodesmata are found dispersed in the CW, while in the case of giant cells, a large amount of them is formed between the walls of giant cells and on the walls that surround them (^{Hofmann et al., 2010}). This suggests the existence of a massive solute transportation system via symplast (^{Hofmann et al., 2010}; ^{Vieira et al., 2012}). In addition, the flow of nutrients to and from the giant cells can also be mediated by specialized transport proteins located in the membrane (^{Rodiuc et al., 2014}).

From the beginning of the formation of the syncytium, the dissolution of cell walls, alterations in their configuration, and increases in their syntheses are evident. These changes are necessary for both the formation of invaginations in areas near the xylem and for the thickening of the CW that surrounds the syncytium (^{Golinowski et al., 1996}; ^{Rodiuc et al., 2014}); since although the cell walls that make up the syncytium break, the cell walls that confine it extend and thicken to resist the increase in pressure created inside the SFS (^{Golinowski et al., 1996}). In addition, the deposition of new material obstructs the existing plasmodesmata and some neighboring cells divide and fuse and others differentiate in the new xylem (vessels) and phloem (sieve tube elements) tissues (^{Hoth et al., 2008}; ^{Rodiuc et al., 2014}). As in giant cells, during the conformation of the syncytium, CW invaginations takes place near the xylem and phloem, which are elongated, branched, and form sophisticated reticulations that expand apically, making the basal parts of the invaginations fuse and wide CW thicknesses form. However, these invaginations are only evident 5 to 7 days after the infection occurs, once the process of SEA formation has advanced (^{Golinowski et al., 1996}). It seems that in the syncytia the formation of invaginations in the CW is a secondary reaction, unrelated to the differenciation of the syncytium and could be caused by an increase in the flow of solutes to the SFS (^{Golinowski et al., 1996}; ^{Rodiuc et al., 2014}).

The CW of immature syncytia thickens in a uniform manner, except in sections of the wall that are in direct contact with the sieve tubes, the walls of which remain thin until the neighboring walls thicken (^{Grundler et al., 1998}). Plasmodesmata are important points of initial degradation of cell walls, and are therefore essential for the formation and expansion of the syncytium (^{Hofmann et al., 2010}); in early stages of the syncytium formation, due to the temporary deposition on callose, nutrients are transported from the phloem apoplastically through transmembrane transporters (^{Hofmann and Grundler, 2006}; ^{Rodiuc et al., 2014}); between 4 and 7 days after innoculation (dai),the amount of plasmodesmata increases and the deposition of callose decreases (^{Hofmann and Grundler, 2006}), and in later stages, after 10 dai, the syncytia connect through the plasmodesmata, making the transfer of nutrients possible (^{Hofmann and Grundler, 2006}; ^{Hofmann et al., 2010}).

The modifications that take place during the formation of the SFS could be the result of coordinated alterations induced by the nematode through the manipulation of the expression of host genes that codify for proteins such as extensins (EXT), expansins (α- y β expansinas), pectin acetylesterases (PAE), pectate lyases (PEL), and endoglucanases (endo-β-1,4-glucanasas), which participate in the modification of cell walls in giant cells, whereas in syncytia it is attributed to the participation of genes that codify for expansins (α- and β expansins), endoglucanases (endo-β-1,4-glucanasas), EXT, polygalacturonases (PG) y pectin acetilasas (PE) (^{Rodiuc et al., 2014}).

Modifications in the synthesis and accumulation of lignin in the SFS

SFS are the only food source for sedentary endoparasitic nematodes and are essential for their growth and reproduction, which is why nematodes, through the induction of extensive changes in the gene expression, must include complex changes in the morphology, metabolism, and physiology of host cells, for their formation. SFS are found in the vascular cylinder, ensuring the necessary contact with the xylem and phloem to provide the SFS with nutrients, therefore alterations in the morphology, thickness, and composition of the CW are a necessary requirement for the formation of the SFS, and therefore, for the successful establishment of sedentary endoparasitic nematodes.

The effectors, synthesized in the oesophageal glands and secreted through the stylet of nematodes, induce the de-differentiation and re- differentiation of the root cells in SFS , although the identification of these secretions is limited (^{Mitchum et al., 2013}). It is known that of the three oesophageal glands, the two subventrals have a greater activity during the invasion of the root and the migration of the nematodes in early stages, whereas the dorsal gland increases its activity during the formation and manintenance of the SFS, that is, in the nematode´s sedentary stage (^{Davis et al., 2008}; ^{Mitchum et al., 2013}).

Nematodes such as Rotylenchulus spp., Tylenchulus spp., Nacobbus spp., and Xiphinema spp. Also induce the formation of SFS in the roots of hosts, although they have been studied little in comparison to those induced by the root-knot nematodes Meloidogyne spp. (giant cells) and those that form cysts (Globodera spp. and Heterodera spp., syncytiums). Both types of SFS share some structural characteristics, yet their ontogeny is different (^{Jones and Northcote, 1972}; ^{Rodiuc et al., 2014}). Root-knot nematodes induce the formation of giant cells when the J2 are hosted in the area of differentiation of the vascular cylinder and each individual induces the differentiation of five to seven parenchymal cells in giant multinuclear cells (^{Abad et al., 2009}). The syncytium, in turn, is formed when the J2 are hosted near the vascular cylinder and from only one cell begins the progressive thickening or narrowing of the plasmodesmata, until the cell from which the SFS formation began fuses with neighboring cells by the CW dissolution to form a large multinuclear syncytium (^{Turner and Rowe, 2006}). In both feeding sites there is an increase in metabolic activity and cytoplasmic density, numerous small vacuoles, proliferation of organelles, particularly of the Golgi apparatus, mitochondria, plastids, ribosomes, and endoplasmic reticula (^{Rodiuc et al., 2014}).

Nematode infection promots the synthesis of phenylpropanoids compounds (^{Edens et al., 1995}; ^{Balbridge et al., 1998}). The induction of lignin biosynthesis occurs as a response to biotic and abiotic stress and is considered an important defense mechanism during the plant-pathogen interaction. Although it is true that this induction will occur in every plant-nematode interaction, it is also true that the regulation of its synthesis will vary, depending on whether the interaction is compatible or incompatible. In order for the restructuring of host cells to take place during the formation of the SFS, changes in the expression in a large number of genes are crucial. In this regard, ^{Jammes et al. (2005)} carried out a global analysis of the A. thaliana transcriptome during its compatible interaction with M. incognita and found that out of 22089 genes monitored, 15% displayed a differential expression during the development of giant cells; the expression of genes involved in the regulation of the cell cycle, DNA processing, protein synthesis and energy presented high levels of expression, as well as the expression of genes related to the metabolism of the cell wall that codify for pectate lyases, expansins, glycoside hyrdolases, xyloglucan endotransglycosylases and one glucose oxidase; in contrast, two genes related to defense, patatin and a protein similar to germin, turned out to be the most strongly repressed; however, they do not report the monitoring of genes specifically involved in the biosynthesis of lignin.

^{Ithal et al. (2007)} reported that in soya plants innoculated with the nematode Heterodera glycines the activity of genes implied in the pathway of the phenylpropanoids, favoring the synthesis of a wide variety of secondary metabolites in plants, such as flavonoids and anthocyanins, and lignin and suberin, which make up the cell walls. The accumulation of phytoalexins, the deposition of lignin and the accumulation of phenolic compounds are characteristic of the defense response in plants; however, the functions of these secondary metabolites in the plant-nematode interactions are not entirely clear, since although its overexpression could be a part of the response of the plant to the infection by the nematodes in an incompatible interaction, these components could play another part in a compatible interaction (^{Ithal et al., 2007}).

During the development of syncytia, the extensive thickening of its cell walls takes place, and although the composition of the new material deposited has not been characterized, histological studies show that the center of the external cortical walls, as well as the center of some fragments of cell wall inside the syncytia, become dyed with tolonium chloride, indicating the presence of lignin and some polyphenoles (^{Jones and Northcote, 1972}). In this way, the increase in the synthesis and accumulation of lignin, not only becomes part of the plant's defense against infection by nematodes, but can also contribute to the formation of the cell wall de novo, to help protect and strengthen the syncytium (^{Ithal et al., 2007}). The transcription of POX genes that codify for peroxidases has been reported to increase during the formation of syncytia. Peroxidases not only eliminate the oxygen-reactive species produced typically as a plant's early defense response against a wide variety of pathogens (^{Marrs, 1996}; ^{Blokhina et al., 2003}), but they also seem to be involved in strengthening the cell wall of the syncytium by the crossing of cell wall polymers (^{Schopfer, 1996}; ^{Darley et al., 2001}), or by its contribution to polymerization of extensins and to the crossing of polysaccharides by the dimerization of phenols (^{Darley et al., 2001}).

The fact that most of the genes that codify for enzymes involved in the biosynthesis of lignin (PAL, C4H, C3H, F5H, CCoAOMT, COMT, CCR, and CAD) have been overexpressed in soybean during the formation of syncytium at 2, 5, and 10 days after infection by H. glycines, indicates that these genes also have an important function in the formation of SFS (^{Ithal et al., 2007}). In Arabidopsis thaliana plants that overexpressed the C4H (early enzyme in the pathway of the phenylpropanoids) and F5H (enzyme that catalyzes the irreversible hydroxylation of G precursors toward the biosynthesis of S units) enzymes, in which the production of G units was inhibited and the amount of S units increased by 50%, as compared to the wild plants, the nematode reproduction was significantly reduced (^{Wuyts et al., 2006}). The increase of S lignin in the vascular bundles might had inhibited the flow of nutrients towards the giant cells or impeded nematode feeding (^{Wuyts et al., 2006}). Similarly when the content of S units was reduced by 10% in tobacco plants in which the expression of the enzyme isoflavona O-metiltransferasa (OMT) was blocked, M. incognita completed its life cycle in a shorter time and there was a higher number of juveniles as compared to the plants where OMT was not bocked (^{Wuyts et al., 2006}). In contrast to the above, ^{Quentin et al. (2009)} found that in A. thaliana plants in which the levels of S lignin fell due to the blockage of activity in enzyme COMT, the growth of the nematode and its life cycle were similar to that observed in control plants in which the enzyme was not blocked. However, A. thaliana is, by nature, susceptible to M. incognita, and therefore in this case the differences in the content of lignin monomers (G or S) in the plant tissues did not influence the infection and life cycle of the nematode in A. thaliana, although one cannot exclude the possibility that in other plants, changes in the activity of enzyme COMT can determine the type of interaction (compatible or incompatible) (^{Pegard et al., 2005}).

Based on investigations cited, it is possible to infer that it is very likely that the type and predominance of the monolignols that form the lignin, specifically the type of monomeric unit (S, G, or H) deposited, can influence the infection and reproduction of sedentary endoparasitic nematodes during its interaction with its host, since these nematodes establish their SFS inside the vascular cylinder, where there is an abundant deposition of polymers for the formation of secondary walls and a high lignification of roots.

On the other hand ^{Wuyts et al. (2007)} point out that it is possible for the pathway of phenylpropanoids to be redirected towards the synthesis of compounds related to resistance. In this way, the hydroxycinnamic acids covalently bonded to the polysaccharides of the CW constitute a possible second physical (and chemical) barrier to the nutrition and migration of the nematodes in the cortex of the roots. Given the physiological importance of the products in the pathway of the phenylpropanoids in the plant-sedentary endoparasitic nematode, the pathway seems to be directed, in the new CW synthesis, towards the formation of the monomers that form a type of lignin that favors the formation of functional SFS. As opposed to what occurs in incompatible ones, the pathway is directed towards the biosynthesis of lignin, which is a barrier for physical defense and secondary metabolites with antimicrobial properties.