Physiological Basis of Smut Infectivity in the Early Stages of Sugar Cane Colonization.

Sugar cane smut (Sporisorium scitamineum) interactions have been traditionally considered from the plant's point of view: How can resistant sugar cane plants defend themselves against smut disease? Resistant plants induce several defensive mechanisms that oppose fungal attacks. Herein, an overall view of Sporisorium scitamineum's mechanisms of infection and the defense mechanisms of plants are presented. Quorum sensing effects and a continuous reorganization of cytoskeletal components, where actin, myosin, and microtubules are required to work together, seem to be some of the keys to a successful attack.

Polyamines levels increase in smut teliospores after contact with sugarcane glycoproteins as a plant defensive mechanism.

Previous studies have already highlighted the correlation between Sporisorium scitamineum pathogenicity and sugarcane polyamine accumulation. It was shown that high infectivity correlates with an increase in the amount of spermidine, spermine and cadaverine conjugated to phenols in the sensitive cultivars whereas resistant plants mainly produce free putrescine. However, these previous studies did not clarify the role of these polyamides in the disorders caused to the plant. Therefore, the purpose of this research is to clarify the effect of polyamines on the development of smut disease. In this paper, commercial polyamines were firstly assayed on smut teliospores germination. Secondly, effects were correlated to changes in endogenous polyamines after contact with defense sugarcane glycoproteins. Low concentrations of spermidine significantly activated teliospore germination, while putrescine had no activating effect on germination. Interestingly, it was observed that the diamine caused nuclear decondensation and breakage of the teliospore cell wall whereas the treatment of teliospores with spermidine did not induce nuclear decondensation or cell wall breakdown. Moreover, the number of polymerized microtubules increased in the presence of 7.5 mM spermidine but it decreased with putrescine which indicates that polyamines effects on Sporisorium scitamineum teliospore germination could be mediated through microtubules interaction. An increased production of polyamines in smut teliospores has been related to sugarcane resistance to the disease. Teliospores incubation with high molecular mass glycoproteins (HMMG) from the uninoculated resistant variety of sugarcane, Mayari 55-14, caused an increase of the insoluble fraction of putrescine, spermidine and spermine inside the teliospore cells. Moreover, the level of the soluble fraction of spermidine (S fraction) increased inside teliospores and the excess was released to the medium. The HMMG glycoproteins purified from Mayarí 55-14 plants previously inoculated with the pathogen significantly increased the levels of both retained and secreted soluble putrescine and spermidine. Polyamines levels did not increase in teliospores after incubation with HMMG produced by non resistant variety Barbados 42231 which could be related to the incapacity of these plants to defend themselves against smut disease. Thus, a hypothesis about the role of polyamines in sugarcane-smut interaction is explained.

Phytochemical and biological evaluation of metabolites produced by alginate-immobilized Bionts isolated from the lichen Cladonia substellata vain.

In this work, new biotechnological procedures have been optimized on the basis of immobilization in alginate of bionts isolated from the lichen C. substellata. From these immobilizates, soluble and biologically active phenolics can be obtained. During bionts-immobilization, stictic, norstictic and usnic acids were secreted to the medium. The amount produced of each of them differed depending on the immobilization time, the precursor supplied and the type of biont used. Greater amounts of stictic acid were detected and maintained over time in all bioreactors. The opposite occurs in non-immobilized thallus. Virtually, all lichen phenols exhibit antioxidant activity to a greater or lesser degree, so that the antioxidant capacity of stictic acid (82.13% oxidation inhibition) was tested. The soluble extract of immobilized algae co-incubated in sodium acetate with fungal hyphae contained carbohydrates and exhibited a potent antioxidant capacity after 13 days of immobilization (94.87%). Therefore, attempts have been made to relate both parameters. On the other hand, the growth of Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae was inhibited by phenolic compounds produced by immobilizates, although the organic extract of the whole lichen showed the highest activity due to a possible synergy with other indeterminate compounds. Thus, C. substellata immobilized bionts are a potential source of different natural antioxidant and antimicrobial compounds.

Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia.

This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD.

Fungal lectin of Peltigera canina induces chemotropism of compatible Nostoc cells by constriction-relaxation pulses of cyanobiont cytoskeleton.

A glycosylated arginase acting as a fungal lectin from Peltigera canina is able to produce recruitment of cyanobiont Nostoc cells and their adhesion to the hyphal surface. This implies that the cyanobiont would develop organelles to motility towards the chemoattractant. However when visualized by transmission electron microscopy, Nostoc cells recently isolated from P. canina thallus do not reveal any motile, superficial organelles, although their surface was covered by small spindles and serrated layer related to gliding. The use of S-(3,4-dichlorobenzyl)isothiourea, blebbistatin, phalloidin and latrunculin A provide circumstantial evidence that actin microfilaments rather than MreB, the actin-like protein from prokaryota, and, probably, an ATPase which develops contractile function similar to that of myosin II, are involved in cell motility. These experimental facts, the absence of superficial elements (fimbriae, pili or flagellum) related to cell movement, and the appearance of sunken cells during of 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 act as a chemoattractant.

Sugarcane glycoproteins may act as signals for the production of xanthan in the plant-associated bacterium Xanthomonas albilineans.

Visual symptoms of leaf scald necrosis in sugarcane (Saccharum officinarum) leaves develop in parallel to the accumulation of a fibrous material invading exocellular spaces and both xylem and phloem. These fibers are produced and secreted by the plant-associated bacterium Xanthomonas albilineans. Electron microscopy and specific staining methods for polysaccharides reveal the polysaccharidic nature of this material. These polysaccharides are not present in healthy leaves or in those from diseased plants without visual symptoms of leaf scald. Bacteria in several leaf tissues have been detected by immunogold labelling. The bacterial polysaccharide is not produced in axenic culture but it is actively synthesized when the microbes invade the host plant. This finding may be due to the production of plant glycoproteins after bacteria infection, which inhibit microbial proteases. In summary, our data are consistent with the existence of a positive feedback loop in which plant-produced glycoproteins act as a cell-to-bacteria signal that promotes xanthan production, by protecting some enzymes of xanthan biosynthesis against from bacterial proteolytic degradation. 

Isolation and characterization of a cyanobacterium-binding protein and its cell wall receptor in the lichen Peltigera canina.

Peltigera canina, a cyanolichen containing Nostoc as cyanobiont, produces and secretes arginase to a medium containing arginine. Secreted arginase acts as a lectin by binding to the surface of Nostoc cells through a specific receptor which develops urease activity. The enzyme urease has been located in the cell wall of recently isolated cyanobionts. Cytochemical detection of urease is achieved by producing a black, electron-dense precipitate of cobalt sulfide proceeding from CO(2) evolved from urea hydrolysis in the presence of cobalt chloride. This urease has been pre-purified by affinity chromatography on a bead of active agarose to which arginase was attached. Urease was eluted from the beads by 50 mM alpha-D-galactose. The experimentally probed fact that a fungal lectin developing subsidiary arginase activity acts as a recognition factor of compatible algal cells in chlorolichens can now been expanded to cyanolichens.

Xanthan production by Xanthomonas albilineans infecting sugarcane stalks.

Xanthomonas albilineans is the causal organism of leaf scald, a bacterial vascular disease of sugarcane. Xanthomonas may invade the parenchyma between the bundles and cause reddened pockets of gum, identified as a xanthan-like polysaccharide. Since xanthan contains glucuronic acid, the ability of Xanthomonas to produce an active UDP glucose dehydrogenase is often seen as a virulence factor. X. albilineans axenically cultured did not secrete xanthans to Willbrink liquid media, but the use of inoculated sugarcane tissues for producing and characterizing xanthans has been required. A hypothesis about the role of sugarcane polysaccharides to assure the production of bacterial xanthan is discussed.

Purification and properties of an unusual UDP-glucose dehydrogenase, NADPH-dependent, from Xanthomonas albilineans.

Xanthomonas albilineans produces a UDP-glucose dehydrogenase growing on sucrose. The enzyme oxidizes UDP-glucose to UDP-glucuronic acid by using molecular oxygen and NADPH. Kinetics of enzymatic oxydation of NADPH is linearly dependent on the amount of oxygen supplied. The enzyme has been purified at homogeneity. The value of pI of the purified enzyme is 8.98 and its molecular mass has been estimated as about 14 kDa. The enzyme shows a michaelian kinetics for UDP-glucose concentrations. The value of K(m) for UDP-glucose is 0.87 mM and 0.26 mM for NADPH, although the enzyme has three different sites to interact with NADPH. The enzyme is inhibited by UDP-glucose concentrations higher than 1.3 mM. N-Terminal sequence has been determined as IQPYNH.

A Lichen Lectin Specifically Binds to the alpha-1,4-Polygalactoside Moiety of Urease Located in the Cell Wall of Homologous Algae.

A lectin from the lichen Evernia prunastri developing arginase activity (EC. 3.5.3.1) binds to the homologous algae that contain polygalactosilated urease (EC. 3.5.1.5) in their cell walls acting as a lectin ligand. The enzyme bound to its ligand shows to be inactive to hydrolyze of arginine. Hydrolysis of the galactoside moiety of urease in intact algae with alpha-1,4-galactosidase (EC. 3.2.1.22) releases high amount of D-galactose and impedes the binding of the lectin to the algal cell wall. However, the use of beta-,4-galactosidase (EC.3.2.1.23) releases low amounts of D-galactose from the algal cell wall and does not change the pattern of binding of the lectin to its ligand. The production of glycosilated urease is restricted to the season in which algal cells divide and this assures the recognition of new phycobiont produced after cell division by its fungal partner.

Antagonism of Gluconacetobacter diazotrophicus (a sugarcane endosymbiont) against Xanthomonas albilineans (pathogen) studied in alginate-immobilized sugarcane stalk tissues.

Xanthomonas albilineans, a pathogenic bacterium that produces leaf scald disease of sugarcane, secretes a xanthan-like gum that invades both xylem and phloem of the host. Xanthan production has been verified after experimental infection of stalk segments of healthy plants. Moreover, Gluconacetobacter diazotrophicus is a nitrogen-fixing endosymbiont of sugarcane plants that antagonizes with X. albilineans by impeding the production of the bacterial gum. The physiological basis of this antagonism has been studied using tissues of sugarcane stalks previously inoculated with the endosymbiont, then immobilized in calcium alginate and maintained in a culture medium for Gluconacetobacter. Under these conditions, bacteria infecting immobilized tissues are able to secrete to the medium a lysozyme-like bacteriocin that inhibits the growth of X. albilineans.

Glycoproteins from sugarcane plants regulate cell polarity of Ustilago scitaminea teliospores.

Saccharum officinarum, cv. Mayarí, is a variety of sugarcane resistant to smut disease caused by Ustilago scitaminea. Sugarcane naturally produces glycoproteins that accumulate in the parenchymatous cells of stalks. These glycoproteins contain a heterofructan as polysaccharide moiety. The concentration of these glycoproteins clearly increases after inoculation of sugarcane plants with smut teliospores, although major symptoms of disease are not observed. These glycoproteins induce homotypic adhesion and inhibit teliospore germination. When glycoproteins from healthy, non-inoculated plants are fractionated, they inhibit actin capping, which occurs before teliospore germination. However, inoculation of smut teliospores induce glycoprotein fractions that promote teliospore polarity and are different from those obtained from healthy plants. These fractions exhibit arginase activity, which is strongly enhanced in inoculated plants. Arginase from healthy plants binds to cell wall teliospores and it is completely desorpted by sucrose, but only 50% of arginase activity from inoculated plants is desorpted by the disaccharide. The data presented herein are consistent with a model of excess arginase entry into teliospores. Arginase synthesized by sugarcane plants as a response to the experimental infection would increase the synthesis of putrescine, which impedes polarization at concentration values higher than 0.05 mM. However, smut teliospores seem to be able to change the pattern of glycoprotein production by sugarcane, thereby promoting the synthesis of different glycoproteins that activate polarization after binding to their cell wall ligand.

Concanavalin A binds to a mannose-containing ligand in the cell wall of some lichen phycobionts.

Concanavalin A, the lectin from Canavalia ensiformis, develops arginase activity depending on Mn(2+). The cation cannot be substituted by Ca(2+) which, in addition, inhibits Mn(2+)-supported activity. Fluorescein-labeled Concanavalin A is able to bind to the cell wall of algal cells recently isolated from Evernia prunastri and Xanthoria parietina thalli. This binding involves a ligand, probably a glycoprotein containing mannose, which can be isolated by affinity chromatography. Analysis by SDS-PAGE reveals that the ligand is a dimeric protein composed by two monomers of 54 and 48 kDa. This ligand shows to be different from the receptor for natural lichen lectins, previously identified as a polygalactosylated urease.

Secreted arginases from phylogenetically farrelated lichen species act as cross-recognition factors for two different algal cells.

Purified arginases secreted from Evernia prunastri and Xanthoria parietina thalli hydrolyze arginine in a Mn2+ -dependent reaction. Ca2+ cannot replace Mn2+, but its addition to reaction mixtures in the presence of Mn2+ significantly inhibited arginase activity. Arginases from both lichen species also show lectin function, binding to the cell wall of both homologous and heterologous algae. Such binding is enhanced by both Ca2+ and Mn2+ and results in cytoagglutination, which is counteracted by alpha-D-galactose. A putative ligand for these lectins consists of a glycosylated urease, the polysaccharide moiety of which is uniquely composed of alpha-D-galactose. Binding of lectins inhibits its enzymatic activity, which is recovered after desorption of the lectin with alpha-D-galactose. Urease is also eluted from arginase-agarose columns by using alpha-D-galactose as eluent. Data demonstrate ligand-dependent retention of the fungal lectin on the algal cell surface and this is consistent with a model of recognition of compatible algae, through which algal cells would form a lichen with a lectin-secreting fungus only when these cells contain the specific ligand for the lectin in their cell walls. This is, lectin binding is used as a mechanism for ensuring specificity in the association.

Gluconacetobacter diazotrophicus, a sugar cane endosymbiont, produces a bacteriocin against Xanthomonas albilineans, a sugar cane pathogen.

Gluconacetobacter diazotrophicus in liquid culture secretes proteins into the medium. Both medium containing Gluconacetobacter protein and a solution of this protein after acetone precipitation appeared to inhibit the growth of Xanthomonas albilineans in solid culture. This apparent inhibition of bacterial growth has, in fact, been revealed to be lysis of bacterial cells, as demonstrated by transmission electron microscopy. Fractionation of the Gluconacetobacter protein mixture in size-exclusion chromatography reveals a main fraction with lysozyme-like activity which produces lysis of both living bacteria and isolated cell walls.

Identification of xanthans isolated from sugarcane juices obtained from scalded plants infected by Xanthomonas albilineans.

The exudate gum produced by Xanthomonas albilineans, a specific sugarcane pathogen, has been isolated from juices of diseased sugarcane stalks, hydrolyzed with hydrochloric acid, and the hydrolysate analyzed by capillary electrophoresis. Sucrose. cellobiose, mannose, glucose, glucose-1-P and glucuronic acid were identified as the major components of the polysaccharide isolated from diseased stalks. Juices from healthy stalks contained maltose instead of cellobiose. The chemical nature of this polysaccharide is discussed.

Production of phenolics by immobilized cells of the lichen Pseudevernia furfuracea: the role of epiphytic bacteria

Immobilized lichen cells from the thalli of the lichen Pseudevernia furfuracea, supplied with acetate as the only source of carbon, continuously produced phenolic substances, atranorin and physodic acid, over 23 days. Epiphytic bacteria associated with the lichen thallus grew actively, probably using both acetate and reduced compounds supplied by lichen cells, since their active growth was avoided by including 10 microM 3,3'-dichlorophenyl-1,1'dimethylurea in the bath solution. Penicillin largely impeded the growth of epiphytic bacteria and decreased phenolic production, which was recovered only at the end of the experimental period, just when the bacteria started a slow, but active growth. We suggest the cooperation of epiphytic bacteria in the biosynthesis of both atranotrin and physodic acid (AU)

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