The polyadenylated RNA of zoospores and growth phase cells of the aquatic fungus,Blastocladiella

The stored poly(A) + RNA from zoospores of the aquatic fungus Blastocladiella emersonii represents 2.5% of the total RNA and has a model MW of 425,000 daltons and an average poly(A) isostich of 32 bases. The poly(A) + RNA also represents 2.5% of the total RNA from early growth phase cells and has a modal MW of 360,000 daltons and an average poly(A) isostich of 38 bases. The poly(A) + RNA from spores and 2-hr plants contains a structure resistant to RNases T₁, T₂, and A, which can be labeled with ³²PO₄ and which will bind to DBAE-cellulose. These characteristics strongly suggest that both the zoospore poly(A) + RNA and the 2-hr cell poly(A) + RNA are capped at the 5′ end; and, hence, it is unlikely that capping is involved in the control of protein synthesis during germination.Approximately 80% of the poly(A) + RNA of the spore is located in the membrane-enclosed ribosomal nuclear cap, and more than 90% of the poly(A) + RNA within the cap is found in the 80S monoribosome and heavier fractions.Synthesis of new poly(A) + RNA occurs very early during zoospore germination, and the labeled poly(A) + RNA rapidly enters the newly organized polysomes. The labeling data for early germination also suggest that cytoplasmic polyadenylation occurs.     

Evidence for differential regulation of two classes of poly(A) RNA inBlastocladiella emersonii zoospores

The poly(A) RNA in zoospores ofBlastocladiella emersonii contains RNA synthesized during the growth phase (GP poly(A) RNA) and late sporulation (LS poly(A) RNA). LS poly(A) RNA synthesized during the final 30 minutes of sporulation is bound exclusively to polyribosomes which comprise approximately 50% of the total zoospore ribosome population. In contrast, GP poly(A) RNA is bound to zoospore monoribosomes. During the final 30 minutes of sporulation, GP poly(A) RNA which is bound to polyribosomes makes a transition to monoribosomes. Zoospore monoribosomes and RNA extracted from zoospore monoribosomes are inactivein vitro while both zoospore polyribosomes and RNA extracted from zoospore polyribosomes stimulate protein synthesis in the wheat germin vitro system. Sedimentation of poly(A) RNA from zoospore monoribosomes on dimethyl sulfoxide gradients revealed that the GP poly(A) RNA was of sufficiently high molecular weight to code for average-sized proteins. These denaturing gradients failed to activate the zoospore monoribosome RNA. The results suggest that the inability to translate zoospore monoribosomesin vitro is due to some property or modification of the zoospore monoribosome poly(A) RNA. Zoospore monoribosomes bound to poly(A) RNA contain an average of two tRNA molecules while zoospore polyribosomes have an average of less than one tRNA bound. This suggests the two classes of ribosomes are blocked at different steps in the elongation process.     

Metabolism of polyadenylic acid sequences during germination of blastocladiella emersoni: Zoospores.

The metabolism of the poly(A) sequences isolated from Blastocladiella emersonii was followed during the first hour of germination. Poly (A) sequences synthesized during the first 30 min of germination do not undergo detectable changes in size. During the first 45 min of germination, poly(A) sequences synthesized during zoosporogenesis decrease in size to the extent that there is essentially no size overlap between poly(A) fragments which were present in the zoospore and newly synthesized poly(A) sequences. The results presented indicate that during germination, polyadenylation occurs in RNA molecules which were present in the zoospore but lacked poly(A) sequences. No detectable size differences were observed between poly(A) sequences added to newly synthesized RNA compared to those added to the nonpolyadenylated RNA present in the zoospore during germination. Cycloheximide did not prevent the observed decrease in size of the poly(A) sequences during germination.     

A temporal analysis of the synthesis of the mRNA sequestered in zoospores of Blastocladiella emersonii

To determine when the dormant mRNA of Blastocladiella emersonii zoospores is synthesized, the metabolism of poly(A) RNA and rRNA was studied during growth and sporulation using pulse-chase techniques. Zoospore poly(A) RNA is synthesized at all stages of the growth cycle investigated in cultures grown either on a normal 15-hr growth cycle or in minicyclic cultures induced to sporulate after only 6.5 hr growth. For cells labeled during the growth phase the specific activity of the pulse-labeled poly(A) RNA and rRNA was identical at the beginning and end of sporulation for any of the 2-hr labeling times investigated. From this it was concluded there is neither a preferential conservation nor degradation during sporulation of the poly(A) RNA and rRNA synthesized at various times during growth. Poly(A) RNA synthesized during early sporulation is preferentially degraded; in contrast, poly(A) RNA synthesized during late sporulation is conserved in the zoospore. Approximately one-third of the total zoospore poly(A) RNA accumulates during the final 15–20 min of sporulation. The accumulation rate for both poly(A) RNA and rRNA decreases as sporulation proceeds. In addition, the rate of degradation for both types of RNA decreases at later stages of sporulation.     

Zoospore adhesion and germination upon non-toxic substances in Pseudulvella sp. (Chlorophyta)

One of the most significant processes in the life history of an alga is the colonization of a new substratum. In the present study, we evaluate whether different organic compounds, such as agar, gelatine, chicken albumin, glycerine and polylysine, promote zoospore recruitment and germination in a periphytic, fresh-water green microalga of the genus Pseudulvella(Chlorophyta). Given the low adhesion capacity of its zoospores a series of experiments were conducted in order to find a substance and its optimal concentration that increases zoospore recruitment and allows us to follow the processes of settlement, attachment and germination of zoospores. Polylysine significantly increased the number of zoospores attached with no significant effect on the germination rate. The minimum effective concentration of polylysine for improving zoospore settlement was 0.1%. %     

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.     

Lipid turnover during morphogenesis in the water mold Blastocladiella emersonii

The lipid content of $\text{Blastocladiella emersonii}$ zoospores is 5 pg/cell or about 13% of dry weight. Within the first few minutes of germination 60–70% of total zoospore lipid is lost, with neutral lipid, glycolipid and phospholipid fractions decreasing to about the same extent. These changes in lipid content precede the breakdown during germination of the complex and extensive membrane system of zoospores. During growth, which immediately follows germination, net phospholipid synthesis resumes so that total lipid is maintained at 6% of dry weight, but net synthesis of neutral and glycolipid does not begin until induction of sporulation. During sporulation the phospholipid level decreases so that the distribution of lipid among the three fractions approaches that found in zoospores. These changes in lipid content suggest that zoospore membranes containing neutral and glycolipids are synthesized $\text{de novo}$ during spore formation.     

Zoospore Homing and Infection Events: Effects of the Biocontrol Bacterium Burkholderia cepacia AMMDR1 on Two Oomycete Pathogens of Pea (Pisum sativum L.)

Burkholderia cepacia AMMDR1 is a biocontrol agent that protects pea and sweet corn seeds from Pythium damping-off in field experiments. The goal of this work was to understand the effect of B. cepacia AMMDR1 on Pythium aphanidermatum and Aphanomyces euteiches zoospore homing events and on infection of pea seeds or roots. In vitro, B. cepacia AMMDR1 caused zoospore lysis, prevented cyst germination, and inhibited germ tube growth of both oomycetes. B. cepacia AMMDR1 also reduced the attractiveness of seed exudates to Pythium zoospores to nondetectable levels. However, when present at high levels on seeds, B. cepacia AMMDR1 had little net effect on zoospore attraction, probably because it also enhanced seed exudation. Seed-applied B. cepacia AMMDR1 dramatically reduced the incidence of infection by Pythium zoospores in situ compared with an antibiosis-deficient Tn5 mutant strain. This mutant strain also decreased Pythium infection incidence to some extent, but only when the pathogen inoculum potential was low. B. cepacia AMMDR1 did not affect attraction of Aphanomyces zoospores or Aphanomyces root rot incidence. These results suggest that B. cepacia AMMDR1 controls P. aphanidermatum largely through antibiosis, but competition for zoospore-attracting compounds can contribute to the effect. Differences in suppression of Aphanomyces and Pythium are discussed in relation to differences in the ecology of the two pathogens.     

Screening capacity of UV-absorbing compounds in spores of Arctic Laminariales

The functional significance of phlorotannins as ultraviolet radiation screens in brown algae is presented. Spectral analysis of zoospore suspensions of the three Arctic Laminariales Saccorhiza dermatodea, Alaria esculenta and Laminaria digitata showed strong absorption in the UV waveband, characteristic of phlorotannins. An induction in the synthesis of the UV-absorbing compound in zoospore suspensions of S. dermatodea and A. esculenta was observed as an increase in absorbance in the UV region after 8 h exposure to the whole light spectrum. Transmission of UVR was also negatively correlated with zoospore density in both these species but not in L. digitata. ‘Biofilters’ constructed from UV-transparent acrylic sheet, containing zoospore suspensions or solutions of phloroglucinol showed varying capacity to protect zoospore cultures from the lethal effects of ultraviolet radiation. Phloroglucinol protects the zoospores from damage by screening out the much harmful shorter UV-B spectra (280-290 nm). Cultured spores of A. esculenta and L. digitata after exposure to the whole light spectrum covered by filters containing phloroglucinol showed high rates of germination, unlike controls covered by seawater-only filters that showed 100% mortality. Biofilters containing zoospore suspensions act as buffers and showed variable UV-protection properties on the germination of its conspecies. At highest zoospore density (∼ 4 × 10⁶ spores ml^− 1), zoospores were observed to screen UV radiation maintaining viability among shielded spores in all species investigated. The protective function of zoospore film is, however, density-dependent in L. digitata. At lower spore density, UV-screening function in S. dermatodea and A. esculenta is attributed to their capacity to accumulate and release UV-absorbing compounds into the medium. Ultraviolet radiation transmission by zoospore suspensions of Saccorhiza and Alaria decreased during exposure to the whole light spectrum which is consistent with the earlier observation of enlarged phenolic vesicles following UVR exposure. The increase in vesicle size and the corresponding increase in UV-absorbing capacity may contribute to greater tolerance of UVR exposure in both species.     

Different life cycle strategies of the dinoflagellates Fragilidium duplocampanaeforme and its prey Dinophysis acuminata may explain their different susceptibilities to the infection by the parasite Parvilucifera infectans

Some marine dinoflagellates form ecdysal cyst (=temporary cysts) as part of their life cycle or under unfavorable growth conditions. Whether the dinoflagellates form ecdysal cysts or not may influence susceptibility to parasitism. In this study, parasite prevalence relative to inoculum size of the parasitoid Parvilucifera infectans zoospores for two dinoflagellate hosts (i.e., Fragilidium duplocampanaeforme and Dinophysis acuminata), which have different life cycle strategies, was examined. Further, susceptibility of cysts to parasitism, encystment signal, duration of encystments, and effects of induced encystment on diel periodicity, using ecdysal cyst-forming F. duplocampanaeforme were explored. The percent hosts infected by P. infectans plotted as a function of inoculum size showed a sharp increase to a maximum in D. acuminata, but a gradual linear rise in F. duplocampanaeforme: while the parasite prevalence in D. acuminata increased to a maximum of 78.8 (±2.4%) by a zoospore:host ratio of 20:1, it in F. duplocampanaeforme only reached 8.9 (±0.3%), even at a zoospore:host ratio of 120:1. In F. duplocampanaeforme, infections were observed only in the vegetative cells and not observed in ecdysal cysts. When exposed to live, frozen, and sonicated zoospores and zoospore filtrate, F. duplocampanaeforme formed ecdysal cysts only when exposed to live zoospores, suggesting that temporary cyst formation in the dinoflagellate resulted from direct contact with zoospores. When the Parvilucifera zoospores attacked and struggled to penetrate F. duplocampanaeforme through its flagellar pore, the Fragilidium cell shed all thecal plates, forming a ‘thecal cloud layer’, in which the zoospores were caught and immobilized and thus could not penetrate anymore. The duration (35 ± 1.8 h) of ecdysal cysts induced with addition of zoospores was significantly longer than that (15 ± 0.8 h) of normally formed cysts (i.e., without addition of zoospores), thereby resulting in delayed growth as well as influencing the pattern of diel periodicity. The results from this study suggest that in addition to the classical predator-prey interaction and allelopathic interaction, parasitism and its accompanying defense can make the food web dynamics much more complicated than previously thought.     

The last two pathway-specific enzyme activities of hexosamine biosynthesis are present in Blastocladiella emersonii zoospores prior to germination

Forward direction assays have been developed for the last two pathway-specific enzymes of hexosamine biosynthesis using crude extracts from Blastocladiella emersonii zoospores. The specific enzyme activities measured are substantially higher than those reported with enzyme preparations from other organisms. During the development of one of the assays, another enzyme activity was observed which converts one of the intermediates of the pathway, N-acetylglucosamine-6-phosphate, to N-acetylglucosamine. The finding of these three enzyme activities in zoospore extracts completes the demonstration that all the enzyme activities necessary to synthesize some 2% by weight as chitin early during zoospore germination (de novo cell wall formation) pre-exist in the zoospore. This demonstration is consistent with the conclusion that the hexosamine pathway is regulated at the post-translational level during zoospore germination.     

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.     

Evidence that Blastocladiella emersonii zoospore chitin synthetase is located at the plasma membrane

Blastocladiella emersonii zoospores are not encased by a cell wall and do not detectably synthesize or contain chitin; accompanying de novo cell wall formation during zoospore encystment, chitin rapidly accumulates and is incorporated into the cell wall. Essential for understanding this abrupt change in chitin synthesis is the location of zoospore chitin synthetase. The enzyme has previously been reported to the sequestered with distinctive cytoplasmic organelles (gamma particles) characteristic for the zoospore cell type. Using similar differential and equilibrium density centrifugation procedures to those reported previously, we have observed the vast majority of zoospore homogenate chitin synthetase activity in fractions distinct from the gamma particle-enriched fractions. Over 90% of the homogenate enzyme activity could be recovered in a sucrose buoyant density region (1.14–1.18 g/ml) containing membranous elements and well separated from the region enriched for gamma particles (1.30–1.34 g/ml). When zoospores were surface-labelled with [³H]concanavalin A prior to homogenization, the buoyant density regions of radioactivity and of chitin synthetase activity exhibited nearly complete coincidence. At least the bulk of zoospore chitin synthetase appears to be located at the plasma membrane, rather than in gamma particles.     

Characterization of RNA synthesized during germination of Blastocladia ramosa zoospores

The synthesis of RNA was studied during the synchronous germination of Blastocladia ramosa zoospores. Comparison of RNA synthesis during germination of B. ramosa and Blastocladiella emersonii zoospores revealed that B. ramosa has a longer lag time before RNA synthesis is initiated and, in addition, the rate of RNA synthesis is ten-fold lower in B. ramosa. Zoospores of B. ramosa were shown to contain pre-formed messenger RNA but this messenger RNA directs only a portion of the protein synthesis which occurs during early germination. The conclusion that the remainder of the protein synthetic activity of the germinating spores is due to new message synthesis was supported by demonstrating that the timing of the initation of protein synthesis on new messages correlates with the time RNA synthesis is initiated. New message synthesis was also demonstrated by the incorporation of label into RNA which contains a poly (A) fragment. Synthesis of all classes of RNA including ribosomal, messenger, and transfer RNA was shown to be initiated at the same time. The implications of this observation are discussed.     

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.     

Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)     

In vitro production of zoospores by the mosquito pathogen Lagenidium giganteum (Oomycetes: Lagenidiales) on solid media

Reliable, large-scale production of Lagenidium giganteum zoospores was obtained on solid media. The fungus was grown for 7 days in a liquid medium of wheat germ, hemp seed, yeast extract, and glucose, then placed onto hemp-seed agar. Zoosporogenesis was induced on agar by immersing the fungal cultures into water. Zoospore production began 10 hr postimmersion, peaked at 18 hr, and ceased by 36 hr. A single, 10-cm Petri dish of fungus on hemp-seed agar produced 1.7?3.8 × 10⁷ zoospores during the 26 hr of zoosporogenesis. Optimal zoospore production occurred with 4- to 7-day-old cultures; cultures older than 10 days produced few zoospores. The temperature range for zoosporogenesis was 15–35°C. The extent of zoosporogenesis was directly related to the volume of water used to induce zoospore formation and inversely proportional to agar thickness. Bioassay of zoospores against second instar Culex quinquefasciatus larvae yielded an LD₅₀ of 400 zoospores/ml.     

Pattern formation of the coenobial algae Pediastrum biwae Negoro

It is suggested that flat colony patterns of the coenobial green algae Pediastrum biwae Negoro can be determined only by some properties of the zoospores without any other control system as a whole. These suggestions are made through observations of colony patterns and colony formation during asexual reproduction and digital electronic computer simulation. The zoospore can be regarded as a sphere which has two C-sites (presumptive sites for connection) and one H-site (presumptive site for horn formation) on its equator. This sphere swims rapidly at random in a transparent vesicle and undergoes a series of changes in its properties: (i) the regions along the equator might come to have a slight affinity for each other, which could cause the arrangement of the zoospores into a plane; (ii) the two C-sites might become connection sites and the zoospores, therefore, might form strings, leading to the characteristic pattern of the adult colony (e.g. a few concentric circular strings, a spiral string and so on); and finally, (iii) the H-site of the zoospore determines whether it grows to a horn or not, i.e. the zoospore grows into a horn cell or a triangular cell according to the absence or presence of other zoospores which prevent its growth.     

The occurrence and distribution of poly(a) ribonucleic Acid in soybean

The occurrence and distribution of poly(A) sequences in the RNA of soybean (Glycine max var. Wayne) have been studied. Only one of the two species of AMP-rich RNA contains poly(A). D-RNA does not contain detectable poly(A) sequences. The TB-RNA is the poly(A) RNA in this system. At least a part (up to 50% or more) of the mRNA in polyribosomes contains a poly(A) sequence. The poly(A) RNA is heterodisperse in size but has a mean size of approximately 18S (2,000 nucleotides) in urea and formamide gels. The poly(A) fragment resulting from ribonuclease A and T₁ digestion migrates as a broad band overlapping the 4 to 5.8S regions of the gels with a mean size of somewhat greater than 5S. No evidence was found for the occurrence of a discrete oligo(A) fragment in the poly(A) RNA; however, oligonucleotides which migrate faster than the poly(A) fraction were observed in preparations which were not bound to oligo(dT) cellulose prior to electrophoresis. This oligonucleotide region was enriched in AMP (up to about 65%) as would be expected after ribonuclease A and T₁ digestion.