Transplantation and subsequent behavior of mitochondria in cells of Phytophthora

Mitochondria isolated from streptomycin-resistant (S(r)) protoplasts of Phytophthora parasitica were transferred into chloramphenicol-resistant (Cpr) protoplasts of P. parasitica or Phytophthora capsici with an average successful rate of 1.7 x 10(-4), using a selective medium containing streptomycin. No colonies appeared when self-fusion products of donor mitochondria or recipient protoplasts were exposed to the selective medium. Mitochondria isolated from Cpr protoplasts of P. capsici were also transferred into S(r) protoplasts of P. parasitica with a similar success rate using a selective medium containing chloramphenicol. Zoospores produced by the Cpr + S(r) intraspecific mitochondrial hybrid gave rise to S(r) and Cpr + S(r) cultures. The second generation zoospores produced by S(r) and Cpr + S(r) cultures also gave rise to S(r) and Cpr + S(r) cultures, suggesting the possible occurrence of fusion between some of the Cpr mitochondria and S(r) mitochondria, and the displacement of non-fused Cpr mitochondria in the receptor protoplast by the donor S(r) mitochondria. Zoospores produced by the interspecific mitochondrial hybrid gave rise to Cpr, S(r), Cpr + S(r), and Cps + Ss cultures. The second generation zoospores produced by Cpr + S(r) or S(r) cultures also gave rise to the same four types of cultures, suggesting the existence of residual antibiotic-sensitive mitochondria (Cps + Ss) in the parental isolates and the random distribution of Cpr, S(r), and Cps + Ss mitochondria during asexual reproduction. Results suggest that the phenotype of antibiotic resistance/sensitivity was the end result of the interactions among the three types of mitochondria.     

Evidence for mitochondrial gene control of mating types in Phytophthora

When protoplasts carrying metalaxyl-resistant (Mr) nuclei from the A1 isolate of Phytophthora parasitica were fused with protoplasts carrying chloroneb-resistant (Cnr) nuclei from the A2 isolate of the same species, fusion products carrying Mr nuclei were either the A2 or A1A2 type, while those carrying Cnr nuclei were the A1, A2, or A1A2 type. Fusion products carrying Mr and Cnr nuclei also behaved as the A1, A2, or A1A2 type. The result refutes the hypothesis that mating types in Phytophthora are controlled by nuclear genes. When nuclei from the A1 isolate of P. parasitica were fused with protoplasts from the A2 isolate of the same species and vice versa, all of the nuclear hybrids expressed the mating type characteristics of the protoplast parent. The same was true when the nuclei from the A1 isolate of P. parasitica were fused with the protoplasts from the A0 isolate of Phytophthora capsici and vice versa. These results confirm the observation that mating type genes are not located in the nuclei and suggest the presence of mating type genes in the cytoplasms of the recipient protoplasts. When mitochondria from the A1 isolate of P. parasitica were fused with protoplasts from the A2 isolate of the same species, the mating type of three out of five regenerated protoplasts was changed to the A1 type. The result demonstrated the decisive effect of mitochondrial donor sexuality on mating type characteristics of mitochondrial hybrids and suggested the presence of mating type genes in mitochondria. All of the mitochondrial hybrids resulting from the transfer of mitochondria from the A0 isolate of P. capsici into protoplasts from the A1 isolate of P. parasitica were all of the A0 type. The result supports the hypothesis of the presence of mating type genes in mitochondria in Phytophthora.     

Creation of hybrid vigor through nuclear transplantation in Phytophthora

When isolated nuclei of a diploid oomycete, Phytophthora parasitica, were fused with protoplasts of another strain of the same species, the regenerated nuclear hybrids grew faster than the parental isolates. Such a phenomenon did not occur in hybrids regenerated from mitochondrion-protoplast or protoplast-protoplast fusion products between these two strains. These results indicate that hybrid vigor is the result of the interaction between two different kinds of nuclei, but not between mitochondria, and they suggest that the presence of mitochondria from nuclear donor cells represses the expression of increased vigor. The nuclear hybrids also expressed increased fungicide resistance and propagule production. Increased vigor in growth was also observed in the interspecific nuclear hybrids when isolated nuclei of P. parasitica were transferred into protoplasts of Phytophthora capsici, and vice versa. This phenomenon may have potential applications, such as the creation of superior fungal strains and plant cultivars with improved commercial traits for usage in industry and agriculture.     

The inheritance of diastatic power and alpha-amylase contents in spring barley

Summary Nuclei were isolated from various types of donor protoplasts and were transferred into receptor protoplasts in numerous combinations. Five percent uptake was achieved under conditions which did not interfere with viability and subsequent culture of receptor protoplasts. Methodological investigations on nuclei uptake were carried out with cereal and tobacco protoplasts. To look for biological proof of integration and replication of transferred nuclear genes, two complementing, chlorophyll-deficient, light-sensitive mutants of tobacco were used as sources of nuclei and receptor protoplasts. Ca. 5.5 × 10⁷ receptor protoplasts were cultured following transplantation experiments involving these complementing mutants and about 1.8 × 10⁷ of the resulting calli were subjected to selective conditions which discriminate against the parental types. No nuclear hybrids were detected, although in control experiments somatic hybrids were obtained by protoplast fusion. Some explanations for failure of nuclear hybrid formation are discussed together with other possible approaches for selective somatic combination of plant cell genophores.     

Karyokinesis in growing and reverting protoplasts ofSchizosaccharomyces pombe

Division of nuclei without cytokinesis proceeds in growing protoplasts ofSchizosaccharomyces pombe. Prior to regeneration of the complete cell wall and reversion the protoplasts contain 1–7 nuclei, protoplasts with 1–2 nuclei are most frequent. When regeneration of the wall is postponed by adding snail enzymes to the growth medium, protoplasts with a higher number of nuclei (2–4) occur. Multinuclear protoplasts can revert to cells. During the first cytokinesis the protoplast with the regenerated cell wall is divided into two cells by a septum, distribution of nuclei between the two cells being probably incidental. More than only a single nucleus can pass to the revertants even during the second cytokinesis. Septation of protoplasts occurs also during a partial blockage of the wall formation by the snail enzyme preparation, however, reversion to cells can never be observed here (it occurs only after transfer of protoplasts to the medium without the enzyme preparation). The growing and reverting protoplasts represent a very good model system for studying relations among individual processes of the cell cycle, primarily growth of the cell, nuclear cycle and cytokinesis. Yeast protoplasts are often utilized as models for studying morphogenic processes, relations among regeneration of the cell wall, including division of the nucleus (karyokinesis) and cytokinesis.     

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.     

Transfer of Isolated Nuclei into Protoplasts of Trichoderma harzianum

Protoplasts released from young hyphae of Trichoderma harzianum contained 0 to 10 nuclei per protoplast, and most (about 80%) contained from 4 to 6 nuclei. Most protoplasts were larger than 3 μm in diameter. Nuclei were isolated from protoplasts of an auxotrophic mutant of T. harzianum and transferred into protoplasts obtained from another auxotroph of the same strain. This intrastrain nuclear transfer gave rise to numerous progeny which were stable, prototrophic, and heterokaryotic. Interstrain transfers in which nuclei from a wild-type prototroph of one strain were transferred into protoplasts from a lysine-deficient auxotroph of a second strain were also done. Heterokaryotic progeny were recovered from these interstrain transfers when the regenerating protoplasts were provided with a low concentration of lysine 48 h after the initial plating. Heterokaryotic progeny contained 11 to 17% of donor-type nuclei. Progeny homokaryotic for donor-type nuclei were obtained as single-spore isolates. These homokaryotic isolates expressed the isozyme pattern and colony morphology phenotype of the nuclear donor. When regenerating protoplasts were provided with lysine 10 days after the initial plating, only a single progeny was obtained. However, single-spore subprogeny of this nuclear transfer were prototrophic and exhibited a wide range of unstable morphological phenotypes.     

TWO TYPES OF CYTOPLASMIC CLEAVAGE IN THE COENOCYTIC SOIL ALGA PROTOSIPHON BOTRYOIDES (CHLOROPHYCEAE)1

Cytokinesis in the coenocytic green alga Protosiphon botryoides (Kütz.) Klebs was studied with transmission electron microscopy. In vegetative cells, nuclei with associated basal bodies and dictyosomes are scattered throughout the cytoplasm. Mature cells may develop either multinucleate resting spores (coenocysts) or uninucleate zoospores. Cytokinesis may be preceded by contraction of the protoplast due to the disintegration of vacuoles that are present in larger, siphonous cells. The formation of coenocysts in ageing, siphonous cells, is signalled by cleavage of the chloroplast and the development of arrays of phycoplast microtubules in one or more transversely oriented planes through the cell. Nuclei with associated basal apparatuses stay dispersed throughout the cytoplasm; the basal bodies apparently are not involved in organization of the phycoplast. The plasma membrane invaginates, resulting in a centripetal cleavage of the protoplast into two or more multinucleate daughter protoplasts. Simultaneously, wall material is deposited along the outside of the daughter protoplasts by dictyosome-derived vesicles, and finally two or more thick-walled coenocysts are formed. The formation of zoospores, on the other hand, is signalled by clustering of the nuclei in one or more groups depending on the shape of the mother cell. The nuclei become arranged with the associated basal apparatuses facing toward the center of the cluster. Bundles of phycoplast microtubules develop between the nuclei, radiating from the center of a cluster toward the plasma membrane; basal apparatuses or associated structures apparently are involved in organization of the phycoplast. Cleavage furrows grow out centrifugally along these bundles of micro-tubules, fed by dictyosome-derived vesicles. No wall material is deposited. An additional mitotic division occurs during cleavage, and finally numerous uninucleate, wall-less, biflagellate zoospores are formed. The ultrastructural features of the two different types of cytoplasmic cleavage associated with two different types of daughter cells have not previously been reported for chlorophycean algae.     

Ultrastructure of nuclei isolated from plant protoplasts

Summary A simple method, involving selective Triton X-100 membrane solubilization, has been developed for the isolation of nuclei from barley and tobacco protoplasts which gives a high yield of essentially pure nuclei. The isolated nuclei resembled those in leaf cells and protoplasts when the isolated nuclei were fixed for short times (2 hours, Medium II), except that their chromatin appeared to be more highly condensed and barley nuclei also lacked the outer nuclear membrane. When longer times of fixation (12 hours, Medium I) were used, the isolated nuclei lacked the characteristic condensed chromatin appearance.     

Transplantation of isolated nuclei into plant protoplasts

The uptake of isolated nuclei from Vicia hajastana Grossh. cells into protoplasts of an auxotrophic cell line of Datura innoxia P. Mill. was induced under the influence of polyethylene glycol and Ca²⁺ at pH 6.8. The frequency of nuclear uptake varied from 0.8 to 2.3% and that of the recovery of prototrophic clones from 10^-5 to 6·10^-4. The prototrophic nuclear fusion products following nuclear uptake could be rescued by initial culture of the protoplasts in non-selective conditions and by the subsequent use of feeder cell layers to support the growth of surviving colonies on a selective medium. The presence of Vicia genomic DNA in some prototrophic clones was confirmed by dot-blot hybridization using Datura and Vicia DNA probes. In certain transformed clones, the recovery of prototrophy was accompanied by the restoration of morphogenetic potential. Welldeveloped shoots typical of wild-type Datura could be regenerated employing an appropriate regeneration medium.Abbreviations MS Murashige and Skoog (1962) - PEG polyethylene glycol     

Levels of polyamines and kinetic characterization of their uptake in the soybean pathogen Phytophthora sojae

Polyamines are ubiquitous biologically active aliphatic cations that are at least transiently available in the soil from decaying organic matter. Our objectives in this study were to characterize polyamine uptake kinetics in Phytophthora sojae zoospores and to quantify endogenous polyamines in hyphae, zoospores, and soybean roots. Zoospores contained 10 times more free putrescine than spermidine, while hyphae contained only 4 times as much free putrescine as spermidine. Zoospores contained no conjugated putrescine, but conjugated spermidine was present. Hyphae contained both conjugated putrescine and spermidine at levels comparable to the hyphal free putrescine and spermidine levels. In soybean roots, cadaverine was the most abundant polyamine, but only putrescine efflux was detected. The selective efflux of putrescine suggests that the regulation of polyamine availability is part of the overall plant strategy to influence microbial growth in the rhizosphere. In zoospores, uptake experiments with [1,4-(14)C]putrescine and [1,4-(14)C]spermidine confirmed the existence of high-affinity polyamine transport for both polyamines. Putrescine uptake was reduced by high levels of exogenous spermidine, but spermidine uptake was not reduced by exogenous putrescine. These observations suggest that P. sojae zoospores express at least two high-affinity polyamine transporters, one that is spermidine specific and a second that is putrescine specific or putrescine preferential. Disruption of polyamine uptake or metabolism has major effects on a wide range of cellular activities in other organisms and has been proposed as a potential control strategy for Phytophthora. Inhibition of polyamine uptake may be a means of reducing the fitness of the zoospore along with subsequent developmental stages that precede infection.     

Transmission genetics of Nicotiana hybrids produced by protoplast fusion

Somatic cell hybrids were produced by fusing protoplasts isolated from callus cells of a tobacco line transformed by Agrobacterium tumefaciens (octopine synthesizing strain B6S3), and mesophyll protoplasts from haploid plants of Nicotiana plumbaginifolia. Hybrids were selected by using differential medium (hormone-independent growth plus greening capacity), or by mechanical isolation and cloning of individual heterokaryocytes. The analysis of hybrid cell lines included the determination of lysopine dehydrogenase activity (encoded by the T-region of Agrobacterium tumefaciens plasmid), examination of isozymes of esterase, and study of chromosome number and morphology. All eight cell lines selected on the screening medium were identified as nuclear hybrids, while only three of the eight evaluated clones obtained by mechanical isolation and cloning were found to be nuclear hydrids; the rest of them were nuclear segregants of tobacco [1] or N. plumbaginifolia [4] type. These data give independent evidence for the occurrence of non-fusion and segregation of nuclei in fusion products, that can be revealed only by using nonselective methods for hybrid screening. In this paper we demonstrate the value of microisolation for the recovery of cytoplasmic hybrids.     

Transfer of isolated nuclei into protoplasts ofSaccharomyces cerevisiae

Cell nuclei were prepared from protoplasts of an adenine-requiring strain ofSaccharomyces cerevisiae, then purified in a discontinuous sucrose gradient, and applied to protoplasts of a recipient strain auxotrophic for uracil, leucine, and histidine. The transfer of the isolated nuclei into protoplasts was induced with polyethylene glycol. The main products of nuclear transfer in young complemented colonies were heterokaryons giving rise to parental type spontaneuos segregants on nutritionally complete medium. After several passages in minimal medium, however, the prototrophic colonies consisted exclusively of stable heterozygous diploid cells.     

Role of ammonia and calcium in lysis of zoospores ofPhytophthora cactorum byBacillus cereus strain UW85

Cultures and cell-free culture filtrates of the biological control agentBacillus cereus strain UW85 lysed zoospores ofPhytophthora cactorum in vitro. Changes in the ionic composition of the growth medium caused by growth of UW85 account for the lytic activity. UW85 raised the pH, excreted ammonia, and removed calcium from the medium during growth and sporulation. Zoospores lysed when pCa²⁺:pNH₃ was greater than 0.8. The lytic activity was produced in uninoculated growth medium by adding ammonium chloride and base to create a pCa²⁺:pNH₃ ratio similar to that of UW85 culture filtrate.     

Establishment and maintenance of nuclear position during zoospore formation in allomyces macrogynus: roles of the cytoskeleton

The involvement of the microtubule (MT) and actin microfilament (MF) cytoskeletons in establishing nuclear positions during zoosporogenesis in Allomyces macrogynus was assessed using selective cytoskeletal disrupting treatments and documented with light microscopy. These experiments were coupled with low-speed centrifugation studies to determine the degree to which cytoskeletal elements anchor nuclear position. At the onset of zoospore formation, nuclei were positioned only in cortical cytoplasmic regions of the zoosporangia (ZS). Immunofluorescence microscopy revealed that MTs primarily emanated from centrosomal regions into the surrounding cytoplasm at this stage. During delimitation of the cytoplasm into individual uninucleate zoospores, nuclei migrated from cortical regions to become distributed throughout the cytoplasm. Coincident with nuclear migrations, MTs were primarily organized at and emanated from nuclear surfaces, forming extensive perinuclear arrays. Nuclear migrations were suppressed in ZS induced to sporulate in the presence of cytochalasin D, an actin MF inhibiting compound. Disruption of MTs with nocodazole did not block nuclear migrations, although resultant nuclear spacing was irregular. Centrifugation treatments of control and drug-treated ZS demonstrated that nuclear positions were stabilized by perinuclear MT arrays. The results indicate that nuclear motility in ZS of A. macrogynus is the result of an actin-based system while perinuclear MTs arrays function to establish and fix nuclear position during zoospore formation. Copyright 1998 Academic Press.     

Recombination after protoplast fusion in the yeast Candida tropicalis

Candida tropicalis protoplasts obtained by snail enzyme treatment were induced to fuse by the use of polyethylene-glycol. Heterokaryons formed by two auxotrophic strains were selected by complementation on minimal medium. These heterokaryons were unstable and readily dissociated into their nuclear components. Under appropriate conditions, the parental nuclei of an heterokaryon fused. The homokaryon so obtained was unstable and segregated into various types of auxotrophic and prototrophic recombinants.List of Abbreviations Used MM minimal medium - YEA yeast extract agar (complete medium) - YPGT yeast-peptone-glucosethiol (medium for protoplast preparation) - PTP medium for cell pretreatment (used before the action of snail enzyme) - PEG polyethylene glycol - p-FPA para-fluorophenylalanine - 5-FC 5-fluorocytosine     

Effect of radiation dosage on efficiency of chloroplast transfer by protoplast fusion in Nicotiana

Chloroplasts of Nicotiana tabacum SR1 were transferred into Nicotiana plumbaginifolia by protoplast fusion. The protoplasts of the organelle donor were irradiated with different lethal doses using a ⁶⁰Co source, to facilitate the elimination of their nuclei from the fusion products. After fusion induction, clones derived from fusion products and containing streptomycin-resistant N. tabacum SR1 chloroplasts were selected by their ability to green on a selective medium. When N. tabacum protoplasts were inactivated by iodoacetate instead of irradiation, the proportion of N. plumbaginifolia nuclear segregant clones was low (1–2%). Irradiation markedly increased this value: Using 50, 120, 210 and 300 J kg^-1 doses, the frequency of segregant clones was 44, 57, 84 and 70 percent, respectively. Regeneration of resistant N. plumbaginifolia plants with SR1 chloroplasts indicated that plastids can be rescued from the irradiated cells by fusion with untreated protoplasts. Resistant N. plumbaginifolia plants that were regenerated (43 clones studied) had diploid (2n = 2X = 20) or tetraploid chromosome numbers and were identical morphologically to parental plants. The absence of aneuploids suggests that in these clones irradiation resulted in complete elimination of the irradiated N. tabacum nuclei. Resistance is inherited maternally (five clones tested). The demonstration of chloroplast transfer and the presence of N. tabacum plastids in the N. plumbaginifolia plants was confirmed by chloroplast DNA fragmentation patterns after EcoRI digestion.     

Somatic hybridization in higher plants.

Somatic hybridization in higher plants has come into focus since methods have been established for protoplast fusion and uptake of foreign DNA and organelles by protoplasts. Polyethylene glycol (PEG) was an effective agent for inducing fusion. Treatment of protoplasts with PEG resulted in 5 to 30% heterospecific fusion products. Protoplasts of different species, genera and even families were compatible when fused. A number of protoplast combinations (soybean + corn, soybean + pea, soybean + tobacco, carrot + barley, etc.) provided fusion products which underwent cell division and callus formation. Fusion products initially were heterokaryocytes. In dividing heterokaryocytes, random distribution of mitotic nuclei was observed to be accompanied by multiple wall formation and to result in chimeral callus. Juxtaposition of mitotic nuclei suggested nuclear fusion and hybrid formation. Fusion of heterospecific interphase nuclei was demonstrated in soybean + pea and carrot + barley heterokaryons. Provided parental protoplasts carry suitable markers, the fusion products can be recognized. For the isolation and cloning of hybrid cells, fusion experiments must be supplemented with a selective system. Complementation of two non-allelic genes that prevent or inhibit growth under special culture conditions appears as the principle on which to base the selection of somatic hybrids. As protoplasts of some species have been induced to regenerate entire plants, the development of hybrid plants from protoplast fusion products is feasible and has already been demonstrated for tobacco.     

一种土壤中大豆疫霉菌分离新方法*

由于腐霉菌的干扰,土壤中大豆疫霉菌的分离十分困难。利用大豆疫霉菌的致病性和大豆对病原菌的选择作用排除腐霉菌,我们建立了一种简单、有效的土壤中大豆疫霉菌的分离方法。该方法用不含抗大豆疫霉根腐病基因的大豆叶碟诱钓大豆疫霉菌的游动孢子,将诱钓叶碟直接接种不含抗大豆疫霉菌基因的大豆植株,再对病株进行选择性或非选择性分离获得大豆疫霉菌。此方法能十分有效地排除腐霉菌干扰和细菌的污染,直接获得纯化菌株。应用该方法我们在以前未报道有大豆疫霉根腐病发生的山东、河南、安徽、江苏和浙江分离到大豆疫霉菌。     

Surface-related Changes of Fusing Phytophthora Zoospores

Zoospores of Phytophthora spp. were fused and then regenerated using a technique previously described. At various points in the fusion and regeneration processes, components of spore surfaces were immunolabelled with specific monoclonal antibodies. Changes in the antigenic properties of spore surfaces during these events were monitored by fluorescence microscopy. Additional, surface-related processes of the phenomena were monitored by electron microscopy, in the presence of lithium ions and polyethylene glycol, zoospores aggregated into clumps and began to fuse. During this time, zoospores retained their reniform shape and antibodies reacted only with plasma membrane-bound antigenic components that are characteristic of the zoosporic state. Immuno-labelling of cell wall occurred only after the fusion solution was replaced with a regeneration solution. As expected, the fusion and the asynchronous regeneration process resulted in the formation of enlarged cells with enlarged or multiple nuclei.