Leucine-lysine synchronization of Allomyces germlings

Summary The leucine-lysine synchronization technique of Dill and Fuller (1970) has been further refined and used to study various biosynthetic events of pre-mitotic germlings of Allomyces neo-moniliformis (the time of DNA replication, RNA synthesis, and protein synthesis), and various morphogenetic changes (germling development, nuclear cap breakdown, and the first mitotic nuclear division). The degree of synchrony induced in a population of germlings appears to be determined by the time when the zoospores are induced to encyst and germinate rather than by the duration of the swimming period of the zoospore. DNA replication, nuclear cap breakdown, early protein synthesis, and morphogenetic development appear to occur prior to messenger RNA synthesis in developing thalli and thus would be under the control of pre-existing messenger RNA. The degree of synchrony of particular morphogenetic or biosynthetic developmental changes induced in a population of A. neo-moniliformis germlings must be determined for each aspect of development which is to be studied.     

A low molecular weight colchicine binding protein from the aquatic Phycomycete Allomyces neo-moniliformis

Summary A colchicine binding protein of the zoospore, flagellum, and pre- and post-mitotic germlings of the aquatic Phycomycete, Allomyces neo-moniliformis, was found to have a sedimentation coefficient of 2.3 S, and a molecular weight of approximately 30 000. The characteristics of this colchicine binding protein from Allomyces are compared to those of the 6 S colchicine binding proteins obtained from a variety of other organisms.     

A novel method for detection of viable zoospores of Pythium in irrigation water

A membrane filtration test has been developed for the detection of viable zoospores of Pythium species. Zoospore suspensions were filtered through 5 (m nitrocellulose membranes and the membranes incubated overnight in 0.07 m glucose, rifamycin (30 mg litre^-1) and pimaricin (100 mg litre^-1). Zoospore germlings were detected using a polyclonal antiserum, raised to mycelial surface washings of five Pythium spp., and visualised with Sigma fast red. The assay gave positive results for all Pythium spp. tested and also to zoospores of Phytophthora cryptogea. Of 10 fungal species isolated from commercial irrigation water, two were detected by the polyclonal antiserum in ELISA tests but only one produced detectable zoospore germlings. The latter isolate was later identified as a Pythium sp. Irrigation water samples collected from commercial UK nurseries yielded zoospores of both Pythium and Phytophthora spp. which, using the assay, were positively identified. Results indicated greater sensitivity than was seen with conventional plating methods. This is a test which could be adapted for on-site use in commercial nurseries.     

Induction of chemiluminescent processes in the fungus Blastocladiella emersonii by exposure to enzyme-generated triplet benzaldehyde

Exposure of the zoospore of Blastocladiella emersonii to the peroxidase-catalyzed aerobic oxidation of phenylacetaldehyde results in: (i) an increased rate of O₂ uptake; (ii) malondialdehyde formation; (iii) light emission with a biphasic kinetics arising from two emitters, one of which is probably a flavin. The process in the zoospore which is responsible for these observations is induced by energy transfer from, or reaction with, the excited species generated in the phenylacetaldehyde/peroxidase/O₂ system prior to lysis. In the case of the germling stage, emission is observed, but no malondialdehyde is formed. Phenylacetaldehyde alone induces a weak emission from non-lysed zoospores or germlings, suggesting in situ excited-state generation.     

Recovery and development of parasexual fusion products inEnteromorpha linza (L.) J. Ag. (Ulvales,Chlorophyta)

Newly released zoospores fromEnteromorpha linza (L.) J. Ag. lack significant cellulose cell wall material and are suitable for treatment as protoplasts in a parasexual fusion process using high pH-Ca^{+ +}, PEG and centrifugation. Treated zoospores settled on glass cover slips within 3 h and were examined microscopically at 1000 ×. Presumptive fusion products were identified by their larger size and presence of twin chloroplasts and eyespots. Unfused zoospores adjacent to fusion cells were killed by 2–3 min exposure to blue light (410–490 nm) from a high pressure mercury illuminator. Unexposed fusion cells developed into uniseriate germlings within 10 days at which stage they could be readily identified at 60 × with a dissecting microscope and isolated by micropipette. Ten-day germlings from both unfused zoospores and fusion cells were stained with the DNA-localizing fluorochrome hydroethidine and relative nuclear DNA content determined with epi-(incident) UV illumination. All germlings were found to be uninucleate. Germlings from unfused zoospores had haploid nuclei with 1N = 10 and 1C and 2C levels of DNA, while germlings from fusion cells had diploid nuclei with 2N = 20 and 2C and 4C levels of DNA. These result are interpreted as evidence of karyogamy following parasexual zoospore fusions. Isolated diploid germlings, cultured for 10 weeks were found to conserve their 2N chromosome complements and elevated levels of nuclear DNA. Although most diploid germlings were morphologically similar to haploid control plants, some exhibited ‘gigas’ characteristics, including larger cells, chloroplasts, and nuclei. These results are discussed in terms of unique phenotypes that result when nuclear and organellar genes are combined in different ways.     

A conditional cell division mutant of Chlamydomonas reinhardii having an increased level of colchicine resistance

Conditional cell division mutants were isolated from Chlamydomonas reinhardii. They were unable to form colonies at 34 °C but not at 23 °C. One of the mutants, TS-60, could neither divide at high nor at low (15 °C) temperature, and seemed to continue protein synthesis at restrictive temperatures. TS-60 also exhibited resistance to 6 mM colchicine which inhibited cell division of the wild-type. Observing that TS-60 flagella were highly resistant to colchicine in their regeneration, it is concluded that the mutational alteration has affected not only the mitotic apparatus but also the flagella. Thermolability of TS-60 was not detected in flagellation but in cell division, though colchicine resistance was expressed in both flagellation and cell division. This suggests that the stable formation of the flagellar microtubule mainly depends on the specific organization of its component. Both thermolability and colchicine resistance of TS-60 were inherited in a Mendelian fashion and unseparable from each other. Reversion tests indicated that the two characters were caused by a single mutation. It is inferred that the above-mentioned phenotypes of TS-60 are the consequence of a mutation in factor(s) involving the colchicine binding activity of tubulin and that this mutational change pleiotrophically leads to some impediment in microtubule formation at restrictive temperature.     

Forecasting infections of the red rot disease on Porphyra yezoensis Ueda (Rhodophyta) cultivation farms

Pythium porphyrae is a fungal pathogen responsible for red rot disease of the seaweed Porphyra (Rhodophyta). Infection forecasts of Porphyra by P. porphyrae were estimated from the epidemiological observations of Porphyra thalli and numbers of zoospore of P. porphyrae in laboratory and cultivation areas. Four features of forecasting infections were determined by relating zoospore concentrations to the incidence of thallus infection; infection (in more than 1000 zoospores L⁻¹), microscopic infection [less than 2 mm in diameter of lesion (in from 2000 to 3000 zoospores L⁻¹)], macroscopic infection [more than 2 mm in diameter of lesion (in from 3000 to 4000 zoospores L⁻¹), and thallus disintegration (in more than 4000 zoospores L⁻¹). High zoospore concentrations led to more infection. The tendency that zoospore concentration of P. porphyrae increased with the rate of infection of Porphyra thalli was generally observed in forecasting infections in both the laboratory and in cultivation areas. Based on the Porphyra cultivation areas, the accuracy and consistency of forecasting infections suggest that this method could be employed to manage and control red rot disease.     

RGS14 is a Microtubule-Associated Protein

Heterotrimeric G-proteins and their regulators are emerging as important players in modulating microtubule polymerization dynamics and in spindle force generation during cell division in C. elegans, D. melanogaster, and mammals. We recently demonstrated that RGS14 is required for completion of the first mitotic division of the mouse embryo, and that it regulates microtubule organization in vivo. Here, we demonstrate that RGS14 is a microtubule associated protein and a component of the mitotic spindle that may regulate microtubule polymerization and spindle organization. Taxol-stabilized tubulin, but not depolymerized tubulin co-immunoprecipitates with RGS14 from cell extracts. Furthermore, RGS14 co-purifies with tubulin from porcine brain following multiple rounds of microtubule polymerization/depolymerization and binds directly to microtubules formed in vitro from pure tubulin (KD=1.3 +/- 0.3 ?M). Both RGS14 and G?i1 in the presence of exogenous GTP promote tubulin polymerization, which is dependent on additional microtubule associated proteins. However, preincubation of RGS14 with G?i1-GDP precludes either from promoting microtubule polymerization, suggesting that a functional GTP/GDP cycle is necessary. Finally, we show that RGS14 is a component of mitotic asters formed in vitro from HeLa cell extracts and that depletion of RGS14 from cell extracts blocks aster formation. Collectively, these results show that RGS14 is a microtubule associated protein that may modulate microtubule dynamics and spindle formation.     

Ultrastructural evidence for the biflagellate origin of the uniflagellate fungal zoospore

Summary The morphological similarities between the kinetosome and the second centriole of the zoospores of Phlyctochytrium kniepii and P. punctatum (Chytridiomycetes) suggest that the second centriole in the chytrid zoospore is a vestigial flagellum base. It is suggested that the term vestigial kinetosome may also be used when referring to the structure which is presently termed the second centriole of the chytrid zoospore. Morphological similarities between the chytrid zoospores of P. kniepii and P. punctatum and the zoospores of Rhizidiomyces apophysatus (Hyphochytridiomycetes) are noted. The possible biflagellate origin of fungi with uniflagellate zoospores is discussed. The third fiber (C fiber) of the kinetosome triplet is shown to form as an outgrowth of the B fiber of the kinetosome doublet.     

RELATIONSHIP OF TEMPERATURE TO ZOOSPORE PRODUCTION IN TETRASPORA GELATINOSA

A strain of Tetraspora gelatinosa was isolated from a field collection and a technique for measuring quantitatively the production of zoospores was devised. The method employed compound colonies grown on agar from zoospore suspensions. The effect of 3 temperatures on zoospore production was tested: no zoospores were produced by the colonies at 3 C; zoospores were produced at 11 and 23 C and the final yield of zoospores at the 2 temperatures was not significantly different. However, at 11 C the maximum density of zoospores produced, measured by cell count, occurred after IS hr, while the maximum density at 23 C occurred after 21 hr.     

Early development in an algal gametophyte: role of the cytoskeleton in germination and nuclear translocation

Summary Biflagellate zoospores from the giant kelpMacrocystis pyrifera underwent germination after adhering to a substrate and produced germ tubes that were approximately 13–15 m in length. Coincident with the germ tube elongation was organelle (other than the nucleus) translocation along the tube. Shortly after formation of the germ tube, the zoospore nucleus divided and one daughter nucleus translocated along the germ tube. The nucleus did not appear to undergo chromosomal condensation prior to division. The nuclear division and/or translocation of the daughter nucleus did not begin until well after germ tube elongation was complete, demonstrating that these are temporally distinct developmental events. The translocation of one daughter nucleus coincided with differentiation of the distal end of the germ tube into a bulbous structure. Following this, the first gametophytic cross wall was formed and, subsequently, the daughter nucleus remaining in the original zoospore body underwent repositioning, assuming a position in the germ tube near the cross wall. Cytochalasin D inhibited germ tube elongation suggesting that actin microfilaments are probably involved in this developmental process. In addition, when cytochalasin D was added to the culture after the germ tube elongation was complete, it did not affect either nuclear division or translocation, indicating that microfilaments were not directly involved in these nuclear events. Colchicine and the plant specific microtubule disrupting agent, amiprophos methyl blocked nuclear division and translocation without affecting germination or germ tube elongation. These data suggest that actin microfilaments are primarily responsible for complete germination, specifically germ tube elongation, while microtubules are involved in nuclear division and translocation. The present study demonstrates that germination (and germ tube elongation) and nuclear translocation inM. pyrifera gametophytes are temporally and mechanistically distinct developmental events.     

Phytophthora nicotianae transformants lacking dynein light chain 1 produce non-flagellate zoospores

Biflagellate zoospores of the highly destructive plant pathogens in the genus Phytophthora are responsible for the initiation of infection of host plants. Zoospore motility is a critical component of the infection process because it allows zoospores to actively target suitable infection sites on potential hosts. Flagellar assembly and function in eukaryotes depends on a number of dynein-based molecular motors that facilitate retrograde intraflagellar transport and sliding of adjacent microtubule doublets in the flagellar axonemes. Dynein light chain 1 (DLC1) is one of a number of proteins in the dynein outer arm multiprotein complex. It is a 22 kDa leucine-rich repeat protein that binds to the catalytic motor domain of the dynein γ heavy chain. We report the cloning and characterization of DLC1 homologues in Phytophthora cinnamomi and Phytophthora nicotianae (PcDLC1 and PnDLC1). PcDLC1 and PnDLC1 are single copy genes that are more highly expressed in sporulating hyphae than in vegetative hyphae, zoospores or germinated cysts. Polyclonal antibodies raised against PnDLC1 locallized PnDLC1 along the length of the flagella of P. nicotianae zoospores. RNAi-mediated silencing of PnDLC1 expression yielded transformants that released non-flagellate, non-motile zoospores from their sporangia. Our observations indicate that zoospore motility is not required for zoospore release from P. nicotianae sporangia or for breakage of the evanescent vesicle into which zoospores are initially discharged.     

Flow cytometric determination of zoospore DNA content and population DNA distribution in cultured Pfiesteria spp. (Pyrrhophyta)

The relative cellular DNA content from 23 different clonal cultures of Pfiesteria spp. zoospores was determined using a DNA fluorochrome and flow cytometry. Significant differences between Pfiesteria piscicida and P. shumwayae were detected, both in mean zoospore DNA content and population cell cycle DNA distribution. Intraspecific differences in DNA content were found between clonal zoospore cultures established from different geographical regions. Long-term cultures (years) of P. piscicida were available for testing, and a negative correlation was observed between zoospore DNA content and time in culture. Zoospore cell cycle-related DNA distributions were also markedly different between the two species in these clonal cultures. In most cultures tested, P. piscicida zoospores exhibited bimodal DNA flow histograms with G1-S-G2+M distributions, typical of eukaryotic asynchronously cycling cells. In contrast, cultures of P. shumwayae zoospores exhibited one DNA peak distribution, indicative of synchronized cells. The data are consistent with the hypothesis that P. shumwayae zoospores are interphasic cells, and mitosis in zoospore cultures of this species predominantly occurs as benthic or adherent non-motile division cysts. Light microscopy observations of the nuclear condition of electrostatically sorted zoospores of each Pfiesteria species also support this hypothesis. If highly conserved, this disparity in modes of vegetative reproduction would ramify the population dynamics of the two Pfiesteria species.     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture I. Some characteristics of the cell cycle

Cells of Chlamydomonas reinhardi Dangeard were grown synchronouslyunder a 12 hr light-12 hr dark regime. Time courses of nucleardivision, chloroplast division, "apparent cytokinesis" and zoosporeliberation were followed during the vegetative cell cycle inthe synchronous culture. Liberation of zoospores occurred atabout 23–24 hr after the beginning of the light periodat 25°C. Four zoospores were produced per mother cell underthe conditions used. At lower temperatures, the process of zoosporeliberation as well as length of the cell cycle was markedlyprolonged, but the number of zoospores produced per mother cellwas approximately the same. At different light intensities,lengths of the cell cycle were virtually the same, while thenumber of zoospores liberated was larger at higher rather thanat lower light intensities. During the dark period, nuclear division, chloroplast divisionand apparent cytokinesis took place, in diis order, and proceededless synchronously than did the process of zoospore liberation.When the 12 hr dark period was replaced with a 12 hr light periodduring one cycle, the time of initiation as well as the durationof zoospore liberation was litde affected in most cases, whereasnuclear division, chloroplast division and apparent cytokinesiswere considerably accelerated by extended illumination. Whenalgal cells which had been exposed to light for 24 hr were furtherincubated in the light, zoospore liberation started much earlierand proceeded far less synchronously, compared with that under12 hr light-12 hr dark alternation. (Received October 12, 1970; )     

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.     

Real-Time Polymerase Chain Reaction Assays for Rapid Detection and Quantification of <Emphasis Type="Italic">Noctiluca scintillans</Emphasis> Zoospore

Application and availability of real-time polymerase chain reaction (PCR) assay to detect and quantify the Noctiluca scintillans zoospore were investigated seasonally. Specific primer set for N. scintillans 18S rDNA was designed and applied to real-time PCR assay using the serial dilutions of N. scintillans zoospores. The real-time PCR assays with Ns63F and Ns260R primers were applied to sea water samples collected weekly in Manazuru Port of Sagami Bay, Japan from April 2005 to June 2006. We developed effective DNA preparation steps for collecting the template DNA of N. scintillans zoospore: size fraction and filter concentration of the water samples, fixation with Lugol solution, cell lysis, and purification. This method is useful for the monitoring of the zoospores of N. scintillans, and can also be used for other small and physiologically fragile planktonic cell. Variation in the density of zoospore was successfully detected in the field samples. The peak density of N. scintillans zoospore was observed to occur just before or at the same time as the peak of the vegetative cells. Moreover, zoospores were detected in seawater even when the vegetative cells were not observed. The presence of zoospore was found all year round in the present study. In this regards, this information is essential for the study of the life cycle and seasonal variation of N. scintillans in the coastal waters.     

The effect of colchicine on colony formation in the algae Hydrodictyon,Pediastrum and Sorastrum

Summary Uninucleate, biflagellate zoospores of Hydrodictyon, Pediastrum and Sorastrum, derived from multinucleate parental cells, aggregate and adhere to form distinctively patterned colonies; earlier work has shown that microtubules underlie the plasmalemma of these zoospores and are also conspicuous in the developing horns of aggregating cells of Pediastrum and Sorastrum. Colchicine applied to parental cells inhibited cytoplasmic cleavage and production of uninucleate zoospores. When zoospores were treated with colchicine, their peripheral microtubules disappeared; the spores failed to aggregate in ordered arrays and did not develop horns. The microtubules therefore appear to play an important role in determining the arrangement of cells in developing colonies by affecting the shape of the cells at the time of their aggregation.     

G protein β subunit is closely associated with microtubules

Previously, we have identified the association of G protein β subunit (Gβ) with mitotic spindles in various mammalian cells. Since microtubules are the main component of mitotic spindles, here we have isolated bovine brain microtubules and purified Gβ subunit to identify the close association of Gβ subunit with purified brain microtubules and have shown the direct incorporation of Gβ subunit into the microtubules both in vitro and in vivo. It was found that: (1) microtubular fraction isolated from bovine brain contained Gβ subunit, (2) coimmunoprecipitation demonstrated that Gβ subunit could be coprecipitated with tubulin, (3) addition of purified Gβ subunit into cytosolic extract for microtubule assembly caused direct incorporation of Gβ subunit into assembled microtubules and increased the association of microtubule-associated proteins with microtubules, and (4) incubation of exogenous Gβ subunit with detergent-permeabilized cells resulted in direct incorporation of Gβ subunit into microtubule fibers and depolymerized tubulin molecules. We conclude that G protein β subunit is closely associated with microtubules and may play an important role in the regulation of microtubule formation in addition to its regulatory role in cellular signal transduction. J. Cell. Biochem. 70:553–562, 1998. © 1998 Wiley-Liss, Inc.