**4.2 Cytoskeleton reorganization in** *S. scitamineum* **cells in response to the binding of sugarcane glycoproteins**

In the early stages of smut disease, spore germination occurs on the internode surface of host stalks, followed by the formation of appressoria, mainly on the inner scale of young buds and on the bases of emerging leaves [49]. Penetration into the plant meristem takes place between 6 and 36 h after fungal cells are deposited on the surface [50]. Since the pathogens normally use the opened stomata of sugarcane leaves to penetrate, it is easy to think that the teliospores deposited at random on the surface of a leaf, far from stomata, should develop a mechanism of displacement toward the way of entry [51]. For this rationale, it is important to demonstrate the existence of these mechanisms and to study how they can be carried out.

Cytoskeleton reorganization in response to the binding of glycoproteins also occurs during *Sporisorium scitamineum*-sugarcane recognition. Moreover, displacement after recognition also results in cytoagglutination of smut teliospores in the same way that activation and chemotaxis of lichen photobionts induced by fungal lectins cause cell aggregation [15]. Interestingly, if glycoproteins are produced by sugarcane-resistant varieties, chemotaxis initially directed to plant invasion results in a "suicide" mechanism.

It has been proposed that at least three classes of glycoproteins exist in the mixture of sugarcane defensive glycoproteins produced by resistant cultivars: (i) a chemotactic glycoprotein, yet uncharacterized; (ii) a cytoagglutinating factor endowed with arginase activity, which also inhibits germination; and (iii) enzymatic proteins that mediate the breakdown of the teliospore cell wall. It has been demonstrated that agglutination of a lot of smut cells in a small region in contact with sugarcane glycoproteins confers resistance, since degradative activity also contained in these glycoproteins (β-1,3-, β-1,4-glucanase, and chitinase) can hydrolyze cell wall of many teliospores at the same time [15]. In this context, it must be pointed out that defensive agglutination depends necessarily on early chemoattraction of cells. For this reason, it is very interesting to go into some depth about how the teliospores movement is stimulated by sugarcane signals. Currently, it has been found that the early chemoattractive effect is fully relevant to trigger a successful defensive response [52]. Lower levels of chemoattractant power exhibited by glycoproteins released by nonresistant cultivars have been directly related to the minor capacity of these plants to defend themselves.

Brand and Gow [53] summarize the knowledge on spore movement in plantpathogen interactions. The two most frequently proposed mechanisms are submicroscopical contractions of helically arranged fibrils within the cell walls and the occurrence of motile appendages in zoospores. Other species of pathogenic fungi produce spores that are capable of gliding in the same way that it occurs for many species of cyanobacteria. Gliding is a form of cell movement that differs from crawling or swimming in which it does not rely on any obvious external organ or change in cell shape and it occurs only in the presence of a substrate [54].

Light and electron microscopy images showed the absence of motile external structures in smut teliospores. However, in the same way that it occurs for *Nostoc*, the invaginations observed during the cellular displacement suggested that cytoskeleton could be the responsible of spore displacement after the contact with sugarcane glycoproteins. Indeed, chemotactic movement of teliospores was strongly inhibited by phalloidin, latrunculin A, and blebbistatin, and the presence of actin and myosin in *S. scitamineum* teliospores has been revealed by immunohistochemical techniques [52].

Teliospores do not need to develop lamellipodia in the direction of movement because they do not "crawl" on a substrate, but "swim" in solution because of the

GDP(β)S, as well as by cyclic AMP [48]. On the contrary, when it is a

noncompatible interaction, lectin penetrates into the cell, promoting putrescine synthesis. The diamine, which causes disorganization of photosynthetic apparatus, activates glucanase that breaks down the cell wall. Compatible and noncompatible

*Scheme of movement of* Nostoc *cells during symbiotic interaction that explains how motility of lichen cyanobionts is due to contraction-relaxation episodes of the cytoskeleton. (1) Chemoattractant lectins released by fungus bind to specific receptors in photobiont cell walls. As a result, the transduction signal that implies cytoskeleton reorganization is activated. (2) Polar cell invaginations are produced by interaction of an ATPase with contractile ability, sensitive to blebbistatin, with F-actin cytoskeleton. (3) After this, depolymerization of F-actin is achieved at the opposite pole, repolymerization of which produces the cell advancement. Representing: , the fungal lectin; , the specific receptor in* Nostoc *cell; , the signal transducing proteins; , the actin monomeres; , the F-actin; , the contractile protein; , the anchorage proteins; , the cell wall; and , the plasmatic membrane. Ferritin-labelled F-actin can be seen in micrographs obtained by*

The absence of superficial elements (fimbriae, pili, or flagellum), related to cell movement, and the appearance of invaginated cells during or after movement, verified by scanning electron microscopy, support the hypothesis that the motility of lichen cyanobionts could be achieved by contraction-relaxation episodes of the cytoskeleton induced by fungal lectin [42]. However, other issues raised included (1) how cytoskeleton is reorganized during migration, (2) how is the mechanism of force generation of movement for cyanobacteria from *P. canina***,** and (3) how it can be related to the invaginations previously observed by electron microscopy. The answers to all of these questions have led to elaborate a proposal of migration

**Figure 6** represents F-actin contraction/relaxing cycles in the *Nostoc* photobiont cells during migration following the lectin gradient. Firstly, binding of arginase molecules to cell wall receptors induces F-actin contraction by means of the activation of a signaling cascade where GTPases must play a main role. At the same time, contraction of filaments must be responsible for invagination appearance in one of the poles of the cell, which is followed by the actin depolarization at the opposite pole. This fact releases the tension from the actin-like cable bound to the membrane, and, finally, induces recovery of the spherical cell shape and movement of

interaction effect on cytoskeleton organization is schematized in **Figure 5**.

mechanism in cyanobacteria.

*transmission electron microscopy (TEM).*

*Parasitology and Microbiology Research*

the cell [42].

**12**

**Figure 6.**

rigidity of the cell wall. Therefore, invagination at the opposite pole would be the only mechanical requirement for cell motion [52]. Again as in *Nostoc* migration, a movement model has been proposed for smut teliospores displacement, which is schemed in **Figure 7**. Firstly, glycoprotein binding to its ligand on cell wall generates a signaling cascade that will trigger cytoskeletal remodeling (1). Translocation of actin filaments has been described as a consequence of the interaction of contractile myosin activity with F-actin cytoskeleton. It leads to an increase in the cell volume at the front of advance and the retraction of the opposite pole. At this pole, filaments must be anchored to cell membrane, since invaginations are observed (2). Finally, depolymerization of F-actin is achieved at the opposite pole, the repolymerization of which leads to cell advancement (3) [30, 52].

to understand, manifest that GTPases should participate in a meticulous regulation

*Role of the Cytoskeletal Actomyosin Complex in the Motility of Cyanobacteria and Fungal Spores*

Moreover, microtubules seem to be also involved in migration mechanism since nocodazole inhibits chemotactic displacement. Interestingly, assays revealed that the negative effect that this drug exerts on chemoattraction is related to a blockage of actin polarization. This demonstrates that actin and microtubules interact, participating together in the establishment of cellular polarity during migration (**Figure 8**). Microtubules-actin interactions regulate important processes in which dynamic cellular asymmetries need to be established such as cell motility, neuronal pathfinding, cellular wound healing, cell division, and cortical flow [57]. The presence of tubulin has also been demonstrated by immunohistochemical techniques in

It is obvious that cytoskeleton reorganization in fungal cells is also involved in germination, in addition to chemotaxis. This is because hyphae of filamentous fungi are very polarized cells and a continuous migration of vesicles from the teliospore cytoplasm through the hyphal cell body to the growing hyphal tip is necessary for organism development [59, 60]. It is clear that cytoskeleton plays a crucial role in polarity establishment in fungal cells during germination: microtubules support nuclei division and long-distance-transport functions in filamentous fungi, whereas actin microfilaments are required for localized targeting events [61]. Microtubule organization in *S. scitamineum* teliospores seems to be crucial for a successful germination [58]. *S. scitamineum* secretes its own arginase, which activates a signal transduction cascade that accelerates teliospore germination when it binds to its cell wall. Moreover, it has been recently suggested that microtubule stabilization during germination of smut teliospores could be triggered by the production of moderate levels of spermidine. *In vitro* microtubule polymerization assays in the presence of spermidine indicate that this polyamine interacts positively with cytoskeleton, probably by means of the positive charges of the molecule. Thus, polyamines are able to bind strongly to existing negative charges in different cellular components, such as nucleic acids, proteins, and phospholipids [62]. Spermidine could act in this

So, polarization of cytoskeleton occurs during both teliospore movement and germination. Herein lies one of the most surprising discover about *S. scitamineum*sugarcane interaction: glycoproteins from resistant to smut plants stimulate the

*Micrographs obtained by fluorescence optical microscope that show F-actin distribution in S. scitamineum cells in the absence (on the left) or in the presence (on the right) of nocodazole (Noc) 0.5 μg.mL-1, an inhibitor of microtubule polymerization. F-actin was detected using phalloidin labeled with fluorescein isothiocyanate (FITC). Red arrows indicate polarized cell. In the middle, percentage of cells with an asymmetric distribution*

of actin organization.

*DOI: http://dx.doi.org/10.5772/intechopen.81299*

*S. scitamineum* cells [58].

**Figure 8.**

**15**

way interacting and stabilizing the cytoskeleton.

*of F-actin in the absence (control) or in the presence of Noc 0.5 μg.mL-1.*

Also in the case of *S. scitamineum* cells, GTPases seem to be relevant in the activation of the movement. GTP causes an enhancement of the inhibition exerted by blebbistatin during migration. However, a large reversion is achieved by addition of GTPγS, the poorly hydrolysable GTP analogue [30, 55] or GDPβS, a deactivator of Rho [56] GTPases, and a total reversion of inhibition can be observed by combining GTP and GTPγS. GTP slightly reverts latrunculin A effect probably at the Rho pathway. However, when GTP analogues, GTPγS and GDPβS are added to the incubation media in the presence of latrunculin A, no reversion of the chemostatic inhibition is observed. These results indicate that GTPγS behaves as a strong activator of the small Rho-GTPase and its downstream pathways, which results in its final switch-off. Conversely, GDPβS blocks this signaling cascade, likely by inhibiting GTP exchange on Rho. These results, that are *not a priori* easy

#### **Figure 7.**

*Scheme of movement of* S. scitamineum *cells during sugarcane-pathogen interaction that explain how motility of teliospores is due to contraction-relaxation episodes of the cytoskeleton. (1) chemoattractant glycoprotein released by sugarcane plants binds to specific receptors in fungal cell walls. As a result, the transduction signal that implies cytoskeleton reorganization is activated. (2) Polar cell invaginations are produced by interaction of myosin II with F-actin cytoskeleton. The translation of the actin filaments into the interior of the cell must begin at its less end, located in the equatorial plane of teliospore. This permits an increase of the cell volume in the front of advance and the retraction of the opposite pole, which produces cell invagination. (3) Repolymerization of F-actin in the front of cell produces the cell advancement. Representing: , the glycoproteins; , the receptor in fungal cell; , the signal transducing proteins; , the actin monomeres; , the F-actin; , the myosin; , the anchorage proteins; , the cell wall; , the plasmatic membrane; and the direction of retrograde flow. F-actin specific labeled ferritin can be seen in micrographs obtained by transmission electron microscopy (TEM).*

*Role of the Cytoskeletal Actomyosin Complex in the Motility of Cyanobacteria and Fungal Spores DOI: http://dx.doi.org/10.5772/intechopen.81299*

to understand, manifest that GTPases should participate in a meticulous regulation of actin organization.

Moreover, microtubules seem to be also involved in migration mechanism since nocodazole inhibits chemotactic displacement. Interestingly, assays revealed that the negative effect that this drug exerts on chemoattraction is related to a blockage of actin polarization. This demonstrates that actin and microtubules interact, participating together in the establishment of cellular polarity during migration (**Figure 8**). Microtubules-actin interactions regulate important processes in which dynamic cellular asymmetries need to be established such as cell motility, neuronal pathfinding, cellular wound healing, cell division, and cortical flow [57]. The presence of tubulin has also been demonstrated by immunohistochemical techniques in *S. scitamineum* cells [58].

It is obvious that cytoskeleton reorganization in fungal cells is also involved in germination, in addition to chemotaxis. This is because hyphae of filamentous fungi are very polarized cells and a continuous migration of vesicles from the teliospore cytoplasm through the hyphal cell body to the growing hyphal tip is necessary for organism development [59, 60]. It is clear that cytoskeleton plays a crucial role in polarity establishment in fungal cells during germination: microtubules support nuclei division and long-distance-transport functions in filamentous fungi, whereas actin microfilaments are required for localized targeting events [61]. Microtubule organization in *S. scitamineum* teliospores seems to be crucial for a successful germination [58]. *S. scitamineum* secretes its own arginase, which activates a signal transduction cascade that accelerates teliospore germination when it binds to its cell wall. Moreover, it has been recently suggested that microtubule stabilization during germination of smut teliospores could be triggered by the production of moderate levels of spermidine. *In vitro* microtubule polymerization assays in the presence of spermidine indicate that this polyamine interacts positively with cytoskeleton, probably by means of the positive charges of the molecule. Thus, polyamines are able to bind strongly to existing negative charges in different cellular components, such as nucleic acids, proteins, and phospholipids [62]. Spermidine could act in this way interacting and stabilizing the cytoskeleton.

So, polarization of cytoskeleton occurs during both teliospore movement and germination. Herein lies one of the most surprising discover about *S. scitamineum*sugarcane interaction: glycoproteins from resistant to smut plants stimulate the

#### **Figure 8.**

rigidity of the cell wall. Therefore, invagination at the opposite pole would be the only mechanical requirement for cell motion [52]. Again as in *Nostoc* migration, a movement model has been proposed for smut teliospores displacement, which is schemed in **Figure 7**. Firstly, glycoprotein binding to its ligand on cell wall generates a signaling cascade that will trigger cytoskeletal remodeling (1). Translocation of actin filaments has been described as a consequence of the interaction of contractile myosin activity with F-actin cytoskeleton. It leads to an increase in the cell volume at the front of advance and the retraction of the opposite pole. At this pole, filaments must be anchored to cell membrane, since invaginations are observed (2).

*Parasitology and Microbiology Research*

Finally, depolymerization of F-actin is achieved at the opposite pole, the repolymerization of which leads to cell advancement (3) [30, 52].

the incubation media in the presence of latrunculin A, no reversion of the

**Figure 7.**

*microscopy (TEM).*

**14**

chemostatic inhibition is observed. These results indicate that GTPγS behaves as a strong activator of the small Rho-GTPase and its downstream pathways, which results in its final switch-off. Conversely, GDPβS blocks this signaling cascade, likely by inhibiting GTP exchange on Rho. These results, that are *not a priori* easy

*Scheme of movement of* S. scitamineum *cells during sugarcane-pathogen interaction that explain how motility of teliospores is due to contraction-relaxation episodes of the cytoskeleton. (1) chemoattractant glycoprotein released by sugarcane plants binds to specific receptors in fungal cell walls. As a result, the transduction signal that implies cytoskeleton reorganization is activated. (2) Polar cell invaginations are produced by interaction of myosin II with F-actin cytoskeleton. The translation of the actin filaments into the interior of the cell must begin at its less end, located in the equatorial plane of teliospore. This permits an increase of the cell volume in the front of advance and the retraction of the opposite pole, which produces cell invagination. (3) Repolymerization of F-actin in the front of cell produces the cell advancement. Representing: , the glycoproteins; , the receptor in fungal cell; , the signal transducing proteins; , the actin monomeres; , the F-actin; , the myosin; , the anchorage proteins; , the cell wall; , the plasmatic membrane; and the direction of retrograde flow. F-actin specific labeled ferritin can be seen in micrographs obtained by transmission electron*

Also in the case of *S. scitamineum* cells, GTPases seem to be relevant in the activation of the movement. GTP causes an enhancement of the inhibition exerted by blebbistatin during migration. However, a large reversion is achieved by addition of GTPγS, the poorly hydrolysable GTP analogue [30, 55] or GDPβS, a deactivator of Rho [56] GTPases, and a total reversion of inhibition can be observed by combining GTP and GTPγS. GTP slightly reverts latrunculin A effect probably at the Rho pathway. However, when GTP analogues, GTPγS and GDPβS are added to

*Micrographs obtained by fluorescence optical microscope that show F-actin distribution in S. scitamineum cells in the absence (on the left) or in the presence (on the right) of nocodazole (Noc) 0.5 μg.mL-1, an inhibitor of microtubule polymerization. F-actin was detected using phalloidin labeled with fluorescein isothiocyanate (FITC). Red arrows indicate polarized cell. In the middle, percentage of cells with an asymmetric distribution of F-actin in the absence (control) or in the presence of Noc 0.5 μg.mL-1.*

organization of the actin cytoskeleton to induce the movement of the teliospores toward the cytoagglutination points but stimulate its depolymerization for avoiding germination. Thus, after displacement as a consequence of cytoskeleton reorganization, the result is agglutinated cells without germinative capacity. Teliospore agglutination without germination triggered by sugarcane arginase becomes the result of a false quorum signal that prevents teliospore infection.
