This study demonstrates the involvement of the cytoskeleton in the movement of cyanobacteria and fungal spores to their hosts to establish a state of symbiosis or pathogenicity. The term symbiosis sensu lato is referred not only to commensalism and mutualism but also to the parasitic aberrations. The establishment of association implies that the endohabitant can move on a wet surface until finding an entry point in the exohabitant surface. In aqueous media, the exohabitant secretes glycoproteins that form a chemoattraction gradient for the invading cells. In lichens, the gradient consists of fungal lectins whose function is to recognize a compatible green alga or cyanobacterium. In the case of pathogens, the secreted proteins usually are a mixture that includes false quorum and chemoattractant signals, and cell wall digestive enzymes. The results indicate that fungal lectins and defense proteins bind to specific cell wall receptors for signaling the activation of cytoskeleton, causing successive cycles of cell contraction-relaxation that permits the migration of the endohabitant. In this study, different biochemical and microscopy techniques have been used. The mechanisms through which the cytoskeleton carries out these cycles of cell contractionrelaxation are described, being this a remarkable advance compared to previous results.
The main interactions between plants include epiphytism, mutualism, commensalism, and parasitism, although the frontier between these types of association can be confusing . For example, most epiphytes do not negatively influence their phytophores since they absorb water and nutrients directly from the atmosphere . It is the case of many bromeliads or the crassulaceae
In this respect, lichens, traditionally considered as an example of mutual symbiosis, exhibit a characteristic that can lead to a decided parasitism: the specificity between symbionts. The fungus selectively chooses individuals from an algal species from its surroundings to form the thallus, while those from other different species will be rejected. This implies that a fungus susceptible to lichenization is able to discriminate between compatible or incompatible algae: the former will form the association while the latter will be eliminated . The argument can be further complicated: if the algae that make up the association split up inside an established thallus, the newly hatched algae may not be recognized as compatible and should therefore be removed (Figure 1D), unless they are able to set up the appropriate recognition systems in time.
Lichen thalli can be reproduced by propagules containing some compatible algal cells surrounded by fungal hyphae. But there is also the possibility that a free-living fungus may find compatible algal cells in its environment. These algae, living in an aqueous film that covers the substrate (soil, rock, tree trunk), can move toward the fungal mass that would envelop them after being recognized. A similar situation is established when a single-cell organism (bacteria, fungal spore) is deposited on the wet surface of a plant, and the higher organism must discriminate whether it is an epiphytic, potential endosymbiont, or decidedly pathogenic microorganism. In the latter two cases, the cells must move in the water film until a suitable point of penetration is found.
Therefore, two main problems arise to explain the mechanisms used to establish this type of interspecific relationship: how unicellular organisms, potential endobionts, move toward the points of contact or entry and how they are recognized by the potential exohabitant when it reaches this position.
Lichens generally secrete glycoproteins to the environment depending on the availability of water . Since most of these glycoproteins were enzymes, it was long time assumed that secretion was a function of the chemical composition of the substrate. This secretion might be taken as a kind of exocellular digestion of the compounds in the medium in order to be internalized into the thallus as simpler structures. However, using the lichen
The aim of this study is to investigate the mechanism by which both prokaryotic and eukaryotic cells that do not have motile organs can move in liquid media thanks to the properties of their actomyosin cytoskeleton.
2. Secreted proteins
In the early stages of the establishment of lichen symbiosis, parasitic attack of the mycobiont (the fungal partner) against a variable number of photobiont cells (algae or cyanobacteria) can occur, which can be attenuated, according to Ahmadjian , by subjecting the neo-association to conditions of deprivation of organic nutrients. In this way, the fungus must keep a vital and active population of green cells, on whose photosynthetic products it depends to maintain its chemoorganic metabolism. This parasitic attack is carried out by invasion of the photosynthetic cells by fungal haustoria or by secretion of proteins that cause changes in genetic expression, structure disorganization, and cell death. These actions require proteins such as arginine methyltransferase, arginase, dioxygenases, or chitinases, according to Joneson et al. , secreted by the fungus
In the case of fungal recognition of an algae considered genetically incompatible, the contact ends with the disorganization of the photosynthetic apparatus and the enzymatic rupture of the cell wall, with the loss of protoplast and death of the cell . When the fungal-secreted arginase does not find a specific receptor in the algal cell wall, the enzyme penetrates the cell wall and activates its own β-1,4-glucanase up to 10 times above its normal physiological level, causing total digestion of specific areas of the cell wall. Such a drastic response contradicts the assertion of Wang et al.  when they state that
Another model of interaction between individuals, studied in our laboratory, is the pathosystem
Therefore, the behavior of the former inhabitant against a process of recognition of compatibility in the symbiosis or defense against a pathogen presents molecular similarities, but a very different characteristic in each case. For lichens, the mycobiont secretes a protein (a lectin) able to discriminate between compatible and incompatible algae . Only in the latter case, the secreted protein behaves as an aggressive factor (Figure 2). In the case of host-pathogen interactions, the proteins secreted by the host are always defense proteins (Figure 3). To carry out these actions, the potential endohabitant, symbiont or pathogen, must possess receptors for the secreted proteins that transmit the signal of compatibility or resistance to the cell machinery when they receive the recognition protein.
The nature of these receptors, both in lichen photobionts as well as in some sugarcane pathogens, has been investigated in our laboratory. The occurrence of a glycosylated urease located in the phycobiont cell wall of
This mode of binding a lectin to the polysaccharide moiety of its ligand by an affinity reaction equals, at the level of action mechanism, the secreted lichen arginases with other, well-known lectins from higher plants, such as concanavalin A (ConA) from
According to this, Marx and Peveling  found that many cultured phycobionts isolated from several lichen species bind to commercial lectins, including Con A and RCA. In addition, Fontaniella et al.  found that ConA is able to bind to the cell wall of algal cells recently isolated from
The binding of sugarcane glycoproteins to their cell wall ligands in the bacterial endophyte
Surprisingly, receptors for both HMMG and MMMG do not behave as the typical adhesion receptors containing polysaccharides that bind by affinity to a specific peptide domain in the signaling molecule, the recognition of which implies the binding of this to selected carbohydrate moieties in their ligands . In this case, the carbohydrate moiety of the signal molecule seems to be used to recognize a particular amino acid domain on the ligand (receptor) in an inverse way to that described for plant lectins and animal selectins. This fact suggests that HMMG and MMMG, with independence of their possible enzymatic activities , behave as true protein of resistance (PR), according to Su et al. , that would require ligands similar to toll-like receptors (TLRs), studied in animals .
The cytoagglutinating effect of sugarcane glycoproteins on smut teliospores was clearly reduced using invertase-digested glycoproteins. This suggested that the hydrolyzed glycidic moiety, which contains fructose residues polymerized as β-D-fructofuranosyl-1,2-β-D-fructose, could be involved in the process of binding since the extensive hydrolysis of β-(1 → 2) bonds impeded cell adhesion. To obtain experimental evidence of the presence of such cell-wall receptor, or receptors, glycoproteins were isolated from the cell wall of the fungal pathogen. These glycoproteins were separated by affinity chromatography through activated agarose columns to which sugarcane glycoproteins from different cultivars had been previously bound. Fungal cell-wall receptors retained by sugarcane glycoproteins were then recovered, desorbed by certain monosaccharides used as eluents . Sugarcane HMMG and MMMG fractions exhibited a high affinity for N-acetyl-D-glucosamine, component of the cell wall of filamentous fungi. Interestingly, this binding mechanism differed, for example, from that described by Blanco et al.  for the cell wall receptors of
4. Cytoskeleton as the main responsible for displacement of
Nostocand Sporisorium scitamineumcells
Directed cell migration is a physical process that involves dramatic modifications in cell shape and, generally, adhesion to the extracellular matrix . Chemoattractive displacement is typically linked to the reorganization of actin filaments in cells, since polarization is the triggering event of cell migration . A ligand on cell surface must activate a signaling pathway that leads to contraction/relaxation of the cytoskeleton. Then, cell polarizes and as a consequence, it moves to the chemoattractant source.
Moreover, many intracellular signaling molecules are involved in cell motility, such as MAPK cascades, lipid kinases, phospholipases, Ser/Thr and Tyr kinases, and scaffold proteins. Specially, GTP molecules play an essential role in both signal transduction and actin organization through Rho GTPases, which appear as the most important components of signaling cascade related to cell migration [38, 39].
Cell migration is the core to modern cell biology. However, progress has been hindered by experimental limitations and the complexity of the process. This has led to the popularity of
4.1 Cytoskeleton reorganization in
Nostoccells in response to the binding of a fungal lectin
For symbiotic interaction, germinating hyphae of the mycobiont needs to meet a compatible photobiont cell, to recognize it, and to make contact . When an isolated fungus and an isolated alga associate, the photobiont migrates toward its potential compatible partner, which implies that the cyanobiont would develop organelles to move toward the fungus. Displacement is particularly relevant in cyanolichens, in which the cyanobiont forms filaments inside the thallus, a segment of which can break off and migrate toward other locations . The recognition process continues during thallus growth, since it is necessary that new generations of photobiont cells become involved in the association .
Lectins found in both prokaryotic and eukaryotic cells play an important role in cell interaction processes. Synthesis of fungal lectins with arginase activity and the occurrence of an algal receptor showing urease activity are absolutely required in the formation of lichen associations . Urease on the algae cell wall acts as a ligand for fungal arginase, fixing it on the cell wall and preventing it to penetrate the cell . So, lectins with arginase activity participate as recognizing proteins of compatible alga binding to a specific receptor on the cell wall. However, they penetrate and cause destruction of algae cells if the specific receptor does not exist . This is the case of noncompatible interaction, as it is shown in Figure 5.
The search for the chemoattractant attracting photobiont cells leads to the discovery of the attractant properties of fungus lectin. In particular, chemotaxis of
However, neither gliding nor blebbing can explain the invaginations observed by electron microscopy in one of the poles of
Some bacterial actin-like proteins or MreB have been already described in free-living cyanobacteria [46, 47] but, contrary to that expected, chemotaxis assays of
Interestingly, when phalloidin was combined with blebbistatin, an eukaryotic myosin II inhibitor, the negative effect on displacement increases (78%), suggesting that blebbistatin may target a molecular target related to chemotaxis in cyanobacteria .
This means that, in the presence of compatible fungus, the binding of the lectin to its specific cell wall receptor would activate the signaling pathway that involves cytoskeleton reorganization. It must take place probably by means of GTPase activity, since the inhibition of chemotaxis produced by the combined action of phalloidin and blebbistatin is largely reversed by GTP and its analogs, GTP(γ)S and GDP(β)S, as well as by cyclic AMP . 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 interaction effect on cytoskeleton organization is schematized in Figure 5.
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 . 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
Figure 6 represents F-actin contraction/relaxing cycles in the
4.2 Cytoskeleton reorganization in
S. scitamineumcells 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 . Penetration into the plant meristem takes place between 6 and 36 h after fungal cells are deposited on the surface . 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 . 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
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 . 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 . 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  summarize the knowledge on spore movement in plant-pathogen 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 .
Light and electron microscopy images showed the absence of motile external structures in smut teliospores. However, in the same way that it occurs for
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 rigidity of the cell wall. Therefore, invagination at the opposite pole would be the only mechanical requirement for cell motion . Again as in
Also in the case of
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 . 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 . Microtubule organization in
So, polarization of cytoskeleton occurs during both teliospore movement and germination. Herein lies one of the most surprising discover about
Cytoskeleton reorganization is the trigger of displacement of
On the other hand, interesting results presented by Díaz et al.  suggest a cytoskeletal-driven mode of cyanobacteria chemotaxis similar to those of eukaryotic cells responding to a chemoattractant gradient. It has been concluded that
F-actin reorganization in response to extracellular chemotactic signaling has been amply studied. Migration is typically linked to the formation of external structures that promote movement. However, similar results in such different systems (lichen and plant pathogen) indicate that this mechanism of cytoskeletal reorganization, which induces cell chemotaxis in absence of lamellipodia/filopodia formation, is conserved in different organisms for recognition between species.