Purification of a naturally produced, low molecular weight organic factor that reversibly blocks encystment of Blastocladiella emersonii zoospores

The water mold Blastocladiella emersonii releases zoospore maintenance factor into the medium during zoosporogenesis. Extracellular factor mediates a reversible developmental block that maintains the motile, cell wall-less zoospore phenotype. A method for purifying the factor is reported that results in 75-120% recovery of biological activity. Analyses of purified factor by thin layer chromatography support the conclusion that factor activity resides in a single organic, low molecular weight molecular species. Other data (Gottschalk, W.K. & Sonneborn, D. R. (1985) J. Biol. Chem. 260, 6592-6599) independently support this conclusion and, in addition, support the conclusion that biological activity resides in an SH-containing cyclic ribotide.     

Blastocladiella emersonii zoospore maintenance factor: A quantitative bioassay used to characterize extracellular appearance and maintenance of factor and interactions between factor,zoospores, and salt

A quantitative bioassay forBlastocladiella emersonii zoospore maintenance factor (ZMF) is documented analyzed. The basis of the assay is an antagonistic interaction between NaCl and ZMF; the former elicits rapid encystment in zoospore populations whereas the latter counteracts this effect. Dilution techniques are used to quantitate the effects of each variable in terms of percentage rapid encystment. Each conditional bioassay variable examined (NaCl concentration, assay pH, cell density, zoospore age, time of assay) appears to affect the sensitivity of zoospore populations to ZMF rather than the level of ZMF activity per se. In particular, zoospore populations “age,” in terms of lowered sensitivity to ZMF, in the following ways: (a) “zoospore aging” without NaCl added and either with or without ZMF added; and (b) “delayed encystment” with NaCl and different fixed levels of ZMF added. ZMF activity in the medium (buffered CaCl₂) accumulates gradually during sporulation; under bioassay conditions and even at subsaturating levels of ZMF activity, activity recoverable from the medium remains stable over long time intervals (4.5–24 h) whether populations remain zoospores or germinate. Populations incubated without added ZMF, whether they remain zoospores or germinate, do not detectably release ZMF activity into the medium. We discuss the following proposals concerning the functions of ZMF: (a) ZMF acts as a negative regulator of themechanics of zoospore encystment; and (b) ZMF acts as a self-generated, natural dispersal signal for the organism.     

Encystment of Pythium aphanidermatum Zoospores is Induced by Root Mucilage Polysaccharides, Pectin and a Monoclonal Antibody to a Surface Antigen

The infection of roots by the pathogenic Oomycete Pythium aphanidermatuminvolves interactions between the fungal zoospores and rootsurface mucilage polysaccharides. After initial recognitionat the root surface the zoospores are triggered to encyst duringwhich adhesive glycoproteins are secreted followed by a fibrillarcyst wall. In this paper a simple in vitro assay has been usedto assess the ability of a variety of macromolecules to inducezoospore encystment. Mucilage polysaccharides of the cress rootsurface trigger encystment. Whole mucilage was fractionatedby gel filtration and a fraction low in uronic acid, containing5% fucose, was shown to be more effective in triggering encystmentthan a uronic acid-rich fraction. Encystment can also be inducedby commercial pectin. The lectin Con A, and PA1, one of a rangeof monoclonal antibodies specific for zoospore surface antigens,also triggered encystment. In Western blotting experiments PA1recognizes protein epitopes of a 75 kDa surface antigen. Theresults suggest that at least one mechanism of zoospore triggeringmay involve a specific zoospore surface receptor. Key words: Pythium aphanidermatum, recognition, encystment, zoospore, mucilage, root, monoclonal antibodies, polysaccharides     

Phosphorylation-dependent regulation of amidotransferase during the development of Blastocladiella emersonii

The enzyme amidotransferase [2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (amino-transferring); EC 2.6.1.16] catalyzes the first step in the hexosamine biosynthetic pathway. In Blastocladiella emersonii the sensitivity of the enzyme to the inhibitor uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) is developmentally regulated. The inhibitable form of amidotransferase activity present in the zoospore is converted to a noninhibitable form during germination. The latter form is present throughout the growth phase and sensitivity to UDP-GlcNAc gradually returns to the zoospore level during sporulation [C.P. Selitrennikoff, N.E. Dalley, and D.R. Sonneborn (1980) Proc. Natl. Acad. Sci. USA 77, 5998-6002]. The following evidence suggests that a phosphorylation/dephosphorylation mechanism underlies this interconversion: (i) Both the vegetative and zoospore enzymes have the same molecular weight of 140,000, but the vegetative enzyme elutes significantly earlier on a DEAE-cellulose column than does the zoospore enzyme. (ii) The increased sensitivity to UDP-GlcNAc occurring in vivo and in vitro correlates with increased phosphorylation of a polypeptide of apparent Mr 76,000. This component copurifies with amidotransferase activity through ion-exchange chromatography and sucrose density gradient centrifugation. (iii) Desensitization and concurrent dephosphorylation of sensitive amidotransferase can be observed in vitro after treatment with a partially purified magnesium-dependent phosphoprotein phosphatase from zoospores.     

Zoospore encystment and pathogenicity of Phytophthora and Pythium species on plant roots

Seven plant species (lucerne, maize, oat, sugarbeet, sorghum, tomato, wheat) and 12 Pythium and Phytophthora species were used in a comparative study designed to investigate the effects of plant and oomycete inter-specific variation on zoospore encystment density and pathogenicity. Zoospores showed differential encystment behaviour and they encysted more on dicotyledonous than on monocotyledonous plants. Pythium aphanidermatum, P. deliense, and Phytophthora nicotianae were the most aggressive species. Sugarbeet was the most severely attacked plant species followed by tomato while oat plants were relatively unaffected. The relationship between zoospore encystment on roots and disease severity depended on the oomycete-plant combination. Correlation analysis between zoospore encystment density and disease severity indicated low and no significant levels (p.05) of association for most plant-oomycete combinations.     

Regulation of attachment, germination, and appressorium formation by zoospores ofLagenidium giganteum and related oomycetes by chitin, chitosan, and catecholamines

Summary Lagenidium giganteum (Oomycetes: Lagenidiales), a facultative parasite of mosquito larvae, infects the larval stage of most species of mosquitoes and a very limited number of alternate hosts. Host infection by this and other members of Oomycetes is initiated by motile, laterally biflagellate zoospores. Chemical bases for the various degrees of host specificity exhibited by these parasites is not known, but presumably involves receptors on the zoospore surface recognizing compounds either secreted by or on the surface of their hosts. Surface topography had no detectable effect onL. giganteum encystment or appressorium formation. Scanning electron microscopy documented the detachment of flagella during zoospore encystment. Bulbous knobs at the basal end of the detached flagellum were interpreted as encysting zoospores dropping the axoneme and/or the basal body and associated structures to which flagella are attached. Multiple signals appear to be involved in the initial steps ofL. giganteum host invasion. Zoospores of this parasite did not encyst on powdered preparations of chitin or chitosan (deacetylated chitin). Upon dissolution of chitosan in dilute acid followed by drying these solutions to form thin, transparent films, zoospores readily encysted. The degree of reacetylation of these films and the spacing of acetylated and deacetylated residues had no significant effect on zoospore encystment. Zoospores of a strain ofLagenidium myophilum isolated from marine shrimp, that also infects mosquito larvae, encysted on chitosan films. No encystment of spores of the plant parasitePhytophthora capsici was observed on chitin or chitosan films. Simulation of cuticle sclerotization by incubating chitosan films with different catecholamines and tyrosinase significantly reduced zoospore encystment. Zoospores that encysted on chitosan films did not germinate in distilled water. Germination could be induced by adding microgram quantities of bovine serum albumin or proteins secreted by motile zoospores into the water, and to a lesser degree by some amino acids, but not by various cations. Zoospores encysted and germinated on the pupal stage of some mosquito species. Appressoria were occasionally formed, but most subsequently sent out another mycelial branch, apparently without attempting to pierce the pupal cuticle. Methylation of pupal exuviae with ethereal diazomethane or methanol/HCl significantly increased zoospore encystment. Modification of chitin by catecholamines, lipids and protein on the epicuticular larval surface all affected host invasion.Abbreviations BSA bovine serum albumin - CID collision-induced dissociation - DOPA 3,4-dihydroxyphenylalanine - ESI-MS electrospray mass spectrometry - ESI-MS/MS tandem electrospray mass spectrometry - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - WGA wheat germ agglutinin - ZAP zoospore aggregation pheromone     

Amelogenin interacts with cytokeratin-5 in ameloblasts during enamel growth

The enamel protein amelogenin binds to GlcNAc (Ravindranath, R. M. H., Moradian-Oldak, R., and Fincham, A.G. (1999) J. Biol. Chem. 274, 2464-2471) and to the GlcNAc-mimicking peptide (GMp) (Ravindranath, R. M. H., Tam, W., Nguyen, P., and Fincham, A. G. (2000) J. Biol. Chem. 275, 39654-39661). The GMp motif in the N-terminal region of the cytokeratin 14 of ameloblasts binds to trityrosyl motif peptide (ATMP) of amelogenin (Ravindranath, R. M. H., Tam, W., Bringas, P., Santos, V., and Fincham, A. G. (2001) J. Biol. Chem. 276, 36586 - 36597). K14 (Type I) pairs with K5 (Type II) in basal epithelial cells; GlcNAc-acylated K5 is identified in ameloblasts. Dosimetric analysis showed the binding affinity of amelogenin to K5 and to GlcNAc-acylated-positive control, ovalbumin. The specific binding of [3H]ATMP with K5 or ovalbumin was confirmed by Scatchard analysis. [3H]ATMP failed to bind to K5 after removal of GlcNAc. Blocking K5 with ATMP abrogates the K5-amelogenin interaction. K5 failed to bind to ATMP when the third proline was substituted with threonine, as in some cases of human X-linked amelogenesis imperfecta or when tyrosyl residues were substituted with phenylalanine. Confocal laser scan microscopic observations on ameloblasts during postnatal (PN) growth of the teeth showed that the K5-amelogenin complex migrated from the cytoplasm to the periphery (on PN day 1) and accumulated at the apical region on day 3. Secretion of amelogenin commences from day 1. K5, similar to K14, may play a role of chaperone during secretion of amelogenin. Upon secretion of amelogenin, K5 pairs with K14. Pairing of K5 and K14 commences on day 3 and ends on day 9. The pairing of K5 and K14 marks the end of secretion of amelogenin.     

Centrin association with the flagellar apparatus in spores ofPhytophthora cinnamomi

Summary Antibodies raised against the calcium-binding protein centrin, were used to identify and localise centrin containing structures in the flagellar apparatus of zoospores and cysts of the oomycetePhytophthora cinnamomi. Immunoblotting of extracts from zoospores indicates that theP. cinnamomi centrin homologue is a 20 kDa protein. Immunofluorescence microscopy with anti-centrin antibodies reveals labelling in the flagella, the basal body connector and co-localisation along the microtubular R1 root (formerly called AR3) that runs from the right side of the basal body of the anterior flagellum into the anterior of the zoospore close to the ventral surface. The centrin (R1^cen) and tubulin components of the R1 root split into four loops on the right hand side of the ventral groove and rejoin along the left hand side of the groove. The R1 root continues down the left hand side of the zoospore past the basal bodies and parallel to the R4 root. We propose that at least inP. cinnamomi there is no R2 root. Immunogold labelling confirms that centrin is a component of the basal body connector complex. When the zoospores become spherical during encystment, the R1^cen pivots by approximately 90 ° with respect to the nucleus.     

Identification of a purC gene from Streptococcus pneumoniae.

A gene encoding 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase was identified in Streptococcus pneumoniae as a 708-bp segment of the genome encoding a 27,001-Da protein with strong similarity to known PurC proteins. The S. pneumoniae purC gene, found immediately adjacent to the competence induction genes, comAB, was cloned and sequenced. The predicted protein product of purC displayed substantial (> 40%) identity to the entire sequence of the PurC proteins of Bacillus subtilis and Escherichia coli. Function of the S. pneumoniae gene product was demonstrated by complementation of E. coli purC mutations.     

A monoclonal antibody and the lectin wheat germ agglutinin induce zoospore encystment in Pythium porphyrae, a marine microbial pathogen

Pythium porphyrae (Oomycota) is a microbial pathogen which causes red rot disease in the commercially cultivated red seaweed Porphyra. This disease is initiated by the motile zoospores of the fungus, which it has been suggested to recognize and process host specific signals by membrane bound receptors. Monoclonal antibodies (MAbs) were developed against the surface components of zoospores and cysts of this fungus in order to try and identify the putative receptor molecules involved in the zoospore encystment process. Screening of MAbs by immunofluorescence assays has revealed three different patterns of surface epitope binding, while labeling of zoospore and cysts components by FITC-conjugated lectins has identified different carbohydrate moieties. Of the MAbs and lectins tested, MAb 1A3 and wheat germ agglutinin have induced zoospore encystment under in vitro conditions. MAb 1A3 identified a 109 KDa band of a glycoprotein in western blot analysis which could be a putative receptor responsible for the induction of zoospore encystment.     

Identification of specific cucumber necrosis virus coat protein amino acids affecting fungus transmission and zoospore attachment

Cucumber necrosis virus (CNV) is naturally transmitted in the soil by zoospores of the fungal vector Olpidium bornovanus. Successful transmission requires that virus particles attach to the surface of zoospores prior to zoospore encystment on host roots. Mechanically passaged CNV was screened for mutants deficient in fungus transmission. We found six such mutants, exhibiting transmission efficiencies ranging from approximately 14 to 76% of that of wild-type (WT) CNV. Results of in vitro virus-zoospore binding assays show that each mutant binds to zoospores less efficiently than WT CNV (21 to 68%), suggesting that defects in transmission for these mutants are at least partially due to inefficient zoospore binding. Analysis of the structure of the CNV coat protein subunit and trimer indicates that affected amino acids in all of the mutants are located in the shell or protruding domain and that five of six of them are potentially exposed on the surface of the virus particle. In addition, several of the mutated sites, along with a previously identified site in a region of subunit-subunit interaction in the coat protein shell domain (M. A. Robbins, R. D. Reade, and D. M. Rochon, Virology 234:138-146, 1997), are located on the particle quasi-threefold axis, suggesting that this region of the capsid may be important in recognition of a putative zoospore receptor. The individual sites may directly affect attachment to a receptor or could indirectly affect attachment via changes in virion conformation.     

Lysine-21 of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase participates in substrate binding through charge-charge interaction.

Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PD) was isolated in high yield and purified to homogeneity from a newly constructed strain of Escherichia coli which lacks its own glucose 6-phosphate dehydrogenase gene. Lys-21 is one of two lysyl residues in the enzyme previously modified by the affinity labels pyridoxal 5'-phosphate and pyridoxal 5'-diphosphate-5'-adenosine, which are competitive inhibitors of the enzyme with respect to glucose 6-phosphate (LaDine, J.R., Carlow, D., Lee, W.T., Cross, R.L., Flynn, T.G., & Levy, H.R., 1991, J. Biol. Chem. 266, 5558-5562). K21R and K21Q mutants of the enzyme were purified to homogeneity and characterized kinetically to determine the function of Lys-21. Both mutant enzymes showed increased Km-values for glucose 6-phosphate compared to wild-type enzyme: 1.4-fold (NAD-linked reaction) and 2.1-fold (NADP-linked reaction) for the K21R enzyme, and 36-fold (NAD-linked reaction) and 53-fold (NADP-linked reaction) for the K21Q enzyme. The Km for NADP+ was unchanged in both mutant enzymes. The Km for NAD+ was increased 1.5- and 3.2-fold, compared to the wild-type enzyme, in the K21R and K21Q enzymes, respectively. For the K21R enzyme the kcat for the NAD- and NADP-linked reactions was unchanged. The kcat for the K21Q enzyme was increased in the NAD-linked reaction by 26% and decreased by 30% in the NADP-linked reaction from the values for the wild-type enzyme. The data are consistent with Lys-21 participating in the binding of the phosphate group of the substrate to the enzyme via charge-charge interaction.     

The primary and secondary spore cyst of Aphanomyces (Oomycetes, Saprolegniales)

The ultrastructural organization of the primary (1°) and secondary (2°) cysts of Aphanomyces astaci and A. laevis is extremely similar, and similar to that of the 1° and 2° cysts of A. eutekhes as presented earlier by Hoch and Mitchell. Synchronous populations of 2° cysts can be induced by mechanical shock and encystment appears to be essentially instantaneous. The cyst coat–wall appears to be formed extremely rapidly from material from the peripheral vesicles with flocculent content. After encystment the microtubule cytoskeleton found in the zoospore is maintained in the 1° and 2° cyst (i.e. the single microtubules which extend along the pyriform nucleus from the ki–netosomes–centrioles and the bundles of closely appressed microtubules are retained). The peripheral vesicles with granular content found in the zoospore are not seen in the 1° or 2° cyst. Multivesicular bodies and lomasomes are observed in the 1° and 2° cyst which are not found in the zoospore. The peripheral cisternae of the zoospore are lost upon encystment and may be formed from dictyosome–derived vesicles during excystment of the 1° and 2° cyst. The U–body of A. astaci has a paracrystalline content while the U–body of A laevis and A eutekhes has a tubular content. A microbody–lipid body complex (sensu Powell) is found in the 1° and 2° cysts of A laevis but not in A astaci or A eutekhes. The significance of the presence of a microbody–lipid body complex in a biflagellate zoospore is discussed.     

Phage-displayed peptides as developmental agonists for Phytophthora capsici zoospores

As part of its pathogenic life cycle, Phytophthora capsici disperses to plants through a motile zoospore stage. Molecules on the zoospore surface are involved in reception of environmental signals that direct preinfection behavior. We developed a phage display protocol to identify peptides that bind to the surface molecules of P. capsici zoospores in vitro. The selected phage-displayed peptides contained an abundance of polar amino acids and proline but were otherwise not conserved. About half of the selected phage that were tested concomitantly induced zoospore encystment in the absence of other signaling agents. A display phage was shown to bind to the zoospore but not to the cyst form of P. capsici. Two free peptides corresponding to active phage were similarly able to induce encystment of zoospores, indicating that their ability to serve as signaling ligands did not depend on their exact molecular context. Isolation and subsequent expression of peptides that act on pathogens could allow the identification of receptor molecules on the zoospore surface, in addition to forming the basis for a novel plant disease resistance strategy.     

Specific induction of encystment ofLagenidium giganteum zoospores by concanavalin A and derivatives of chitin and chitosan

Summary Zoospores of the mosquito pathogenic fungusLagenidium giganteum preferentially attach to and encyst on the cuticular surface of the immature stages of many species of mosquitoes as the initial step in the infection process. Recognition by zoospores of specific chemical or physical signals on the cuticular surface triggers attachment. A number of compounds likely to be present on the surface of mosquito larvae were evaluated for efficacy in eliciting zoospore encystment. Free amino acids and oligomers, a number of phenolic and polyphenolic compounds and most carbohydrates did not induce encystment at concentrations less than 500 g/ml. Colloidal chitin and chitin films were also ineffective as was O-carboxy-methylchitin; however, glycol chitin and glycol chitosan induced rapid encystment at concentrations at or below 1 g/ml. Zoospores also attached to and encysted in great numbers on fibers of oxycellulose, but not on cellulose. Concanavalin A was the only lectin which induced encystment at concentrations less than 10 g/ml, which suggests that a glycoprotein with terminal mannose and/or glucose residues is involved in encystment. A number of phenols were metabolized by peroxidase on the zoospore surface. Addition of hydrogen peroxide to zoospore suspensions reduced the time needed to induce zoospore encystment by some phenols; however, there was no consistent relationship between the presence or absence of this synergistic effect and the ability ofL. giganteum peroxidase to metabolize a given substrate. The sterol-binding compound amphotericin B induced immediate encystment at 3.5 g/ml, suggesting that sterols, which are required for the induction of zoosporogenesis, were present on the zoospore membrane.     

The ultrastructure of cell wall formation and of gamma particles during encystment of Allomyces macrogynus zoospores

Structural changes during cell wall formation by populations of semisynchronously germinating zoospores were studied in the water mold Allomyces macrogynus. Fluorescence microscopy using Calcofluor white ST (which binds to -1,4-linked glycans) demonstrated that Calcofluor-specific material was deposited around most cells between 2–10 min after the induction of encystment (beginning when a wall-less zoospore retracts its flagellum and rounds up). During the first 15 min of encystment there was a progressive increase in fluorescence intensity. Ultrastructural analysis of encysting cells showed that within 2–10 min after the induction of encystment small vesicles 35–70 nm diameter were present near the spore surface, and some were in the process of fusing with the plasma membrane. The fusion of vesicles with the zoospore membrane was concomitant with the appearance of electron-opaque fibrillar material outside the plasma membrane. Vesicles similar to those near the spore surface were found within the gamma () particles of encysting cells. These particles had a crystalline inclusion within the electron-opaque matrix. During the period of initial cyst cell wall formation numerous vesicles appeared to arise at the crystal-matrix interface. Approximately 15–20 min was required for the cell wall to be formed. We suggest that the initial response of the zoospore to induction of encystment is the formation of a cell wall mediated by the fusion of cytoplasmic vesicles with the plasma membrane.Non-Standard Abbreviations GlcNac N-Acetylglucosamine - DS sterile dilute salts solution - PYG peptone-yeast extract-glucose broth     

Calcium efflux associated with encystment of Phytophthora palmivora zoospores

Zoospores of the fungus $\text{Phytophthora palmivora}$, pre-labeled with ⁴⁵Ca, excreted up to 30% of their total ⁴⁵Ca when stimulated to encyst. Excretion was essentially completed within 90 sec of the application of the stimulus. Encystment of the population was completed within 5 min. Four different stimuli were used: pectin addition (420 μg ml^?1), Sr²⁺ addition (5 mM), cyclic AMP addition (6.7 mM) and mechanical agitation. The kinetics and amount of Ca excretion were essentially the same in each case. The calcium ionophore A23187 increased the rate of ⁴⁵Ca uptake by motile zoospores, incubated in 100 μM CaCl₂, but did not induce encystment under these conditions. The ionophore did not induce ⁴⁵Ca efflux from pre-labeled zoospores. Incubation in EGTA and in K⁺ failed to induce either encystment or ⁴⁵Ca excretion. We conclude that rapid excretion of a significant proportion of the zoospore calcium is linked to the early stage of stimulus-induced encystment, and that this comes from an intracellularly located, non-cytoplasmic source, such as the peripheral vesicles, but that changes in cellular Ca²⁺ are not necessarily the single controlling factor in the induction of encystment.