Production of monoclonal antibodies against peripheral-vesicle proteins in zoospores of Phytophthora nicotianae.

A coimmunisation protocol using microsomal fractions from Phytophthora nicotianae cells has enhanced the production of monoclonal antibodies directed towards proteins produced during asexual sporulation. Over 40% of the antibodies targeted three categories of zoospore peripheral vesicles. Five antibodies label the contents of dorsal vesicles, with three of these reacting with two P. nicotianae polypeptides with a relative molecular mass of approximately 100 kDa. Two antibodies label the contents of large peripheral vesicles and react with two very high-molecular-weight polypeptides in extracts of P. nicotianae cells. These antibodies cross-react with the contents of large peripheral vesicles in P. cinnamomi zoospores. Ten antibodies label the contents of P. nicotianae zoospore ventral vesicles and react with a single polypeptide with a relative molecular mass of 230 kDa. A number of these antibodies against the contents of ventral vesicles in P. nicotianae zoospores cross-react with ventral-vesicle proteins in P. cinnamomi cells in immunofluorescence and immunoblot assays. The study illustrates the value of the coimmunisation protocol and has produced antibodies that could be instrumental in the cloning of genes encoding peripheral-vesicle proteins.

Genes expressed in zoospores of Phytophthora nicotianae.

The genus Phytophthora includes many highly destructive plant pathogens. In many Phytophthora species, pathogen dispersal and initiation of plant infection are achieved by motile, biflagellate zoospores that are chemotactically attracted to suitable infection sites. In order to study gene expression in zoospores, we have constructed a cDNA library using mRNA from zoospores of Phytophthora nicotianae. The library was arrayed and screened using probes derived from mycelium or zoospore mRNA. More than 400 clones representing genes preferentially expressed in zoospores were identified and sequenced from the 5' end of the insert. The expressed sequence tags (ESTs) generated were found to represent 240 genes. The ESTs were compared to sequences in GenBank and in the Phytophthora Genome Consortium database, and classified according to putative function based on homology to known proteins. To further characterize the identified genes, a colony array was created on replicate nylon filters and screened with probes derived from four Phytophthora developmental stages including zoospores, germinating cysts, vegetative mycelium and sporulating hyphae, and from inoculated and uninoculated tobacco seedlings. Data from sequence analysis and colony array screening were compiled into a local database, and searched to identify genes that are preferentially expressed in zoospores for future functional analysis.

Structure and expression of the genes encoding proteins resident in large peripheral vesicles of Phytophthora cinnamomi zoospores.

Zoospores of Phytophthora spp. contain several characteristic types of peripheral vesicles. One of these, large peripheral vesicles, has been proposed to act as a nutrient store and in P. cinnamomi has been shown to contain three immunologically related high-molecular-weight proteins, designated LPVs. We have used antibodies directed against P. cinnamomi zoospores and cysts to isolate several cDNA clones which are products of the Lpv genes and encode one or more of the LPV proteins present in large peripheral vesicles. Northern blot analysis demonstrated the presence of three large Lpv transcripts (11-14 kb) in RNA isolated from hyphae which had been induced to form sporangia. Coordinate accumulation of the three transcripts occurred after induction of sporangium formation: no transcript was observed in uninduced hyphae and maximum transcript levels of all three transcripts were seen 4-6 h after induction. Genomic Southern blots indicated that P. cinnamomi contains three Lpv genes, presumably corresponding to the three transcripts and proteins seen in Northern and Western blots, respectively. Partial genomic clones representing two of the Lpv genes were isolated and characterized by restriction mapping and partial DNA sequencing. In the regions sequenced, the genes were > 99% identical, the high degree of conservation extending at least 415 bp downstream of their polyadenylation sites. The Lpv coding regions contained a variable number (approximately 12-18) of highly conserved 534 bp repeats, flanked by apparently unique sequences. Variation in the number of repeats in the Lpv genes was responsible for the different sizes of the three transcripts and proteins. Database searches using the Lpv nucleotide and deduced amino acid sequences failed to detect any similar sequences. We discuss the molecular events which may have been involved in the evolution of the Lpv genes and the nature of the products of these genes.

Isolation and characterization of four genes encoding pyruvate, phosphate dikinase in the oomycete plant pathogen Phytophthora cinnamomi.

The oomycete genus Phytophthora contains some of the world's most devastating plant pathogens. We report here the existence in P. cinnamomi of four genes encoding the pyrophosphate-utilizing glycolytic/gluconeogenic enzyme pyruvate, phosphate dikinase (PPDK). The coding regions of the four genes are >99% identical. At least three of the genes comprise a small gene cluster, which may have arisen through recent gene duplication and inversion events. Levels of Pdk mRNA are low in vegetative hyphae, but increase rapidly and transiently upon transfer of cultures to nutrient-free media, conditions that trigger asexual sporulation. PPDK protein and enzyme activity levels do not show a similar increase during sporulation. Assays of PPDK activity in P. cinnamomi hyphal extracts suggest that the majority of glycolytic flux in sporulating hyphae probably occurs via PPDK, rather than pyruvate kinase. This finding, combined with the existence of Phytophthora-expressed sequence tags encoding two other pyrophosphate-utilizing enzymes, indicates that pyrophosphate-based metabolism may be important in Phytophthora. The possibility that PPDK and other enzymes of pyrophosphate-based metabolism may provide targets for the development of novel control measures for Phytophthora and other oomycete pathogens is discussed.

Use of molecular cytology to study the structure and biology of phytopathogenic and mycorrhizal fungi.

Molecular cytology, that is, the in situ localization of selected molecules by labeling with lectins, enzymes, and antibodies, has made a major contribution to our understanding of the structure and biology of fungi and is increasingly becoming an integral part of molecular, genetic, and biochemical studies. The review presented in this article concentrates on recent advances in the application of molecular cytology in investigations of the structure and biology of phytopathogenic and mycorrhizal fungi and of the molecular basis of their infection of host plants. The review examines details of the structure and molecular composition of fungal cell walls revealed by lectin, enzyme, and antibody labeling. Molecular composition is shown to vary according to taxonomic relationships and as a reflection of differences in cell type, location within the cell, and within thickness of the wall. Sites of synthesis and secretion of wall components are also detected through the labeling of selected molecules. In situ labeling of cytoskeletal elements, microtubules and actin microfilaments, has provided much information on the role of these elements in tip growth, organelle distribution, and spore development. Molecular cytology, particularly through the generation of monoclonal antibodies, has also revealed new and exciting information on specialized infection structures formed by fungi in order to infect host plants. The sites of storage and secretion of adhesives and degradative enzymes have been documented, as have surface specializations that may be associated with avoidance of detection by the host. In addition, in situ labeling with enzymes and antibodies has aided studies of the host defense response, including mechanisms of detection of fungal elicitor molecules, changes in wall composition, and the secretion of antifungal compounds. With the increasing production of monoclonal antibodies to fungal molecules, molecular cytology promises to continue to make an important contribution to our understanding of fungal cell structure and function in the future.

A transient rise in cytoplasmic free calcium is required to induce cytokinesis in zoosporangia of Phytophthora cinnamomi.

We studied the role of cytoplasmic free Ca2+ concentration ([Ca2+]i) in cytokinesis of zoosporangia of the water mold Phytophthora cinnamomi. In these cells cytokinesis is separated from nuclear division and can be triggered at precisely determined times by cold shock. Changes in [Ca2+]i were monitored by ratiometric fluorescence imaging of pressure microinjected Fura-2 dextran. Two increases in [Ca2+]i always occurred in sporangia that underwent cytokinesis in response to cold shock. Within the first minute of cold shock, [Ca2+]i rose rapidly and transiently to levels 25 to 131% higher than the resting level of 104 +/- 54 nM. By 10 min, [Ca2+]i had decreased and was near the initial resting level. The second increase in [Ca2+]i was gradual and prolonged, accompanying cell division. Near completion of cytokinesis, [Ca2+]i had risen to 231 +/- 165 nM. The initial brief rise in [Ca2+]i was absent in sporangia that did not undergo cleavage. Microinjection of the Ca2+ buffer 5,5'-dibromo-BAPTA before cold shock, blocked cytokinesis suggesting that the transient rise in [Ca2+]i may be necessary for induction. The subsequent gradual increase in [Ca2+]i may not be critical because microinjection of 5,5'-dibromo-BAPTA during cleavage plane development did not always perturb division.

Microtubules regulate the generation of polarity in zoospores of Phytophthora cinnamomi.

Zoospores of Phytophthora cinnamomi are formed by cleavage of a multinucleate sporangium and contain nine different components that are distributed or oriented about a well-defined axis running through a pair of basal bodies near the nucleus. In this study, the importance of the cytoskeleton in establishing and maintaining cellular polarity was examined by using the anti-microtubule drug oryzalin and the anti-microfilament drug cytochalasin D (CD). The effects of the drugs on uncleaved and cleaving sporangia were determined, using fluorescence microscopy, for six of the components that are polarized in untreated cleaved cells: an astral microtubule (MT) array, the nucleus, mitochondria and three different types of vesicles, two of which are involved in directed exocytosis. CD had no effect upon the MT arrays, the positioning of nuclei or the polarized redistribution of mitochondria and vesicles to the cortical cytoplasm, although it did cause abnormal cleavage. The effects of oryzalin, however, indicate that the asymmetric disposition of the MT array is fundamental to zoospore polarities: when the array is itself eliminated with this drug, none of the other five elements show any signs of polar positioning within the cleaved sporangium. Oryzalin also caused abnormal cleavage similar to that seen in CD-treated cells. Most intriguing, however, was the finding that although the three vesicle types in cleaved, oryzalin-treated sporangia did not exhibit the polarized distribution seen in control and CD-treated cells, in many cases the vesicles had, nevertheless, lost their initially random distributions and had become concentrated in the cytoplasm adjacent to the abnormal cleavage planes. Thus although an intact MT array is required for segregation of the vesicles within the cortex, their redistribution to the cortex can somehow occur in the absence of MTs and actin microfilaments.

Freeze substitution reveals a new model for sporangial cleavage in Phytophthora, a result with implications for cytokinesis in other eukaryotes.

Rapid freezing and freeze substitution (RF-FS) have been used to re-examine the process by which the multinucleate sporangium of the Oomycetes, Phytophthora cinnamomi and P. palmivora, is subdivided into uninucleate zoospores. The results indicate a new model for sporangial cleavage in Phytophthora and suggest that the currently accepted model is based on interpretation of artefacts caused by chemical fixation. The previous model describes cleavage as a two-stage process in which specialized cleavage vesicles first become positioned at the boundaries of each future subdivision and later fuse to compartmentalize the sporangium. RF-FS, however, indicates that cleavage results from the progressive extension of paired sheets of membrane along the future subdivision boundaries. These sheets finally interconnect and subdivide the sporangium. Cleavage vesicles are only evident in preliminary stages of this process and are never aligned along the future boundaries, contrary to the observations of studies based on chemical fixation. Chemical fixation apparently causes the membranous sheets to vesiculate, even at relatively advanced stages of cleavage, thus giving the misleading impression that the resulting network of lined-up vesicles is an intermediate stage in the cleavage process. This finding has wide-ranging implications for the understanding of eukaryotic cytokinesis, because all previous studies that describe vesicle alignment and fusion have relied upon chemical fixation. Other novel features revealed by RF-FS include an extensive extracellular matrix within the sporangium that could be involved in zoospore release, and a trans-Golgi network.

Studies on the cell surface of zoospores and cysts of the fungus Phytophthora cinnamomi: The influence of fixation on patterns of lectin binding.

The study of the surface properties of zoospores and cysts of the fungus Phytophthora cinnamomi required a fixation regime that would preserve the cells adequately and not interfere with binding and detection of probes on the cell surface. When they were fixed in 4% formaldehyde (F), specific binding of concanavalin A-fluorescein isothiocyanate and rhodamine-labeled soybean agglutinin was obtained. However, electron microscopy showed that preservation was so poor that intracellular binding sites had become exposed. By contrast glutaraldehyde (G), even at concentrations as low as 0.05%, gave good preservation of the zoospores but induced high levels of nonspecific fluorescence, making its use impractical for studies using fluorescent probes. Addition of 1-4% F to 0.05-0.8% G reduced the level of G-induced fluorescence while not diminishing the quality of ultrastructural preservation. This effect was evaluated quantitatively and an optimum fixation regime for the fungal cells, namely, 0.2% G and 2-4% F in 50 mM PIPES buffer, was determined. This combined fixative facilities correlated fluorescence and ultrastructural labeling with lectins and immunocytochemical probes.

Reorganization of cortical microtubules and cellulose deposition during leaf formation in Graptopetalum paraguayense.

In the "regeneration" of a shoot from a leaf of the succulent, Graptopetalum paraguayense E. Walther the first new organs are leaf primordia. The original arrangement of cellulose microfibrils and of microtubules (MTs) in the epidermis of the leaf-forming site is one of parallel, straight lines. In the new primordium both structures still have a congruent arrangement but it is roughly in the form of concentric circles that surround the new cylindrical organ. The regions which undergo the greatest shift in orientation (90°) were studied in detail. Departures from the original cellulose alignment are detected in changes in the polarized-light image. Departures from the original cortical MT arrangement are detected using electron microscopy. The over-all reorganization of the MT pattern is followed by the tally of MT profiles, the various regions being studied in two perpendicular planes of section. This corrects for the difference in efficiency in counting transverse versus longitudinal profiles of MTs. Reorientation takes place sporadically, cell by cell, for both the cellulose microfibrils and the MTs, indicating a coordinated reorientation of the two structures. That MTs and cellulose microfibrils reorient jointly in individual cells was shown by reconstruction of the arrays of cortical MTs in paradermal sections of individual cells whose recent change in the orientation of cellulose deposition had been detected with polarized light. Closeness of the two alignments was also indicated by images where the MT and microfibril alignments co-varied within a single cell. The change-over in alignment of the MTs appears to involve stages where arrays of contrasting orientation co-exist to give a criss-cross image. During this critical reorganization, the frequency of the MTs is high. It falls during subsequent enlargement of the organ. It was found that the rearrangement of the cortical MTs to approximate a series of concentric circles on the residual meristem occurred before the emergence of leaf primordia. Through their apparent influence on microfibril alignments, the changes in MT disposition, described here, have the potential to generate major biophysical changes that accompany organogenesis.

Interpolation of microtubules into cortical arrays during cell elongation and differentiation in roots of Azolla pinnata.

Longitudinal sections of roots of Azolla pinnata R. Br. were prepared for electron microscopy so that cortical microtubules could be counted along the longitudinal walls in cell files in the endodermis, pericycle, and inner and outer cortex, and in sieve and xylem elements. With the exception of the xylem, where there are no transverse cell divisions, each file of cells commences with its initial cell and then possesses a zone of concomitant cell expansion and transverse cell division, followed, after completion of the divisions, by a zone of terminal cell differentiation. The cells augment their population of cortical microtubules as they elongate and divide, showing a net increase of up to 0.6 micron of polymerized microtubule length per min. Two main sub-processes were found: (i) When a longitudinal wall is first formed it is supplied with a higher number of microtubules per unit length of wall than it will have later, when it is being expanded. This initial quota becomes diluted as the second sub-process commences. (ii) The cells interpolate new microtubules at a rate which is characteristic of the cell, and, in the endodermis, of the face of the cell, while the cell elongates. Most cell types thus maintain a set density of cortical microtubules while they elongate and divide. Comparisons of endodermal cells in untreated controls, and roots that had been treated with colchicine, low temperature, or high pressure indicate that the initial quota of microtubules, and the later interpolations, and differentially sensitive to microtuble perturbations. Three types of behaviour, all related to changes in the cell walls, were noted as cortex, xylem and sieve element cells entered their respective phases of cell differentiation. The cortical cells expanded in all dimensions, and the interpolation of microtubules diminished or ceased. The sieve elements continued to elongate, and interpolated at a high rate, reaching unusually high densities of microtubules when the cell walls were being thickened. During this period a net increase of 2.0 micron of polymerized microtubule length per min was calculated. Thereafter interpolation ceased and the density of microtubules declined. The sample applied to developing xylem except that, because wall-thickening is localized rather than widespread, the rise and subsequent fall in the density of microtubules was less marked. The data are discussed in relation to the participation of microtubules in wall deposition and to the hypothesis that cortical microtubules arise in discrete zones along the edges of cells.

Structure of cortical microtubule arrays in plant cells.

Serial sectioning was used to track the position and measure the lengths of cortical microtubules in glutaraldehyde-osmium tetroxide-fixed root tip cells. Microtubules lying against the longitudinal walls during interphase, those overlying developing xylem thickenings, and those in pre-prophase bands are oriented circumferentially but on average are only about one-eighth of the cell circumference in length, i.e., 2-4 micrometer. The arrays consist of overlapping component microtubules, interconnected by cross bridges where they are grouped and also connected to the plasma membrane. Microtubule lengths vary greatly in any given array, but the probability that any pass right around the cell is extremely low. The majority of the microtubule terminations lie in statistically random positions in the arrays, but nonrandomness in the form of groups of terminations and terminations in short lines parallel to the axis of cell elongation has been observed. Low temperature induces microtubule shortening and increases the frequency of C-shaped terminations over the 1.7% found under normal conditions; colchicine and high pressures produce abnormally large proportions of very short microtubules amongst those that survive the treatments. Deuterium oxide (D2O) treatment probably induces the formation of additional microtubules as distinct from increasing the length of those already present. The distribution of C-shaped terminations provides evidence for at least local polarity in the arrays. The validity of the findings is discussed, along with implications for the development, maintenance, and orientation of the arrays and their possible relationship to the orientation of cellulose deposition.

Formative and proliferative cell divisions, cell differentiation, and developmental changes in the meristem of Azolla roots.

The root of the water fern Azolla is a compact higher-plant organ, advantageous for studies of cell division, cell differentiation, and morphogenesis. The cell complement of A. filiculoides Lam. and A. pinnata R.Br. roots is described, and the lineages of the cell types, all derived ultimately from a tetrahedral apical cell, are characterised in terms of sites and planes of cell division within the formative zone, where the initial cells of the cell files are generated. Subsequent proliferation of the initial cells is highly specific, each cell type having its own programme of divisions prior to terminal differentiation. Both formative and proliferative divisions (but especially the former) occur in regular sequences. Two enantiomorphic forms of root develop, with the dispositions of certain types of cell correlating with the direction, dextrorse or sinistrorse, of the cell-division sequence in the apical cells. Root growth is determinate, the apical cell dividing about 55 times, and its cell-cycle duration decreasing from an initial 10 h to about 4 h during the major phase of root development. Sites of proliferation progress acropetally during aging, but do not penetrate into the zone of formative divisions. The detailed portrait of root development that was obtained is discussed with respect to genetic and epigenetic influences; quantal and non-quantal cell cycles; variation in cell-cycle durations; relationships between cell expansion and cell division: the role of the apical cell; and the limitation of the total number of mitotic cycles during root formation.

Pre-prophase bands of microtubules in all categories of formative and proliferative cell division in Azolla roots.

Pre-prophase bands of microtubules were found in every category of cell division, symmetrical and asymmetrical, in the cell lineages of the root apex of Azolla pinnata R.Br. and A. filiculoides Lam., and in the transverse divisions in the cell files of the roots. They are also found in the asymmetrical cell division that gives rise to trichoblasts in roots of Hydrocharis dubia (B1). Backer. It is possible, in a variety of cell types in roots of Azolla, to predict within a fraction of a micrometre where a new cell wall will be located. In every such case the midline of the 1.5-3-µm-wide pre-prophase band anticipates this location. Each of the daughter cells thus inherits approximately half of the former pre-prophase band site. Images interpreted as stages of formation of the band were obtained, its microtubules replacing the interphase cortical arrays. In one highly asymmetrical division, band formation precedes migration of the nucleus to the site of mitosis. The asymmetrical division that gives rise to root hairs passes acropetally along every cell in the dermatogen layer, and preprophase bands were seen up to 8 cells in advance of the last completed division. Here, and in the zone of formative divisions, the band is present for much longer than the duration of mitosis. The ubiquity of the band in the Azolla root tip is discussed in relation to the literature, and a working hypothesis is presented that takes into account current knowledge of occurrence, development and function of the band.

Evidence for initiation of microtubules in discrete regions of the cell cortex in Azolla root-tip cells, and an hypothesis on the development of cortical arrays of microtubules.

Complexes of microtubules, vesicles, and (to varying degrees) dense matrix material around the microtubules were seen along the edges of cells in root apices of Azolla pinnata R.Br. (viewing the cells as polyhedra with faces, vertices and edges). They are best developed after cytokinesis has been completed, when the daughter cells are reinstating their interphase arrays of microtubules. They are not confined to edges made by the junction of new cell plates with parental walls, but occur also along older edges. Similar matrices and vesicles are seen amongst phragmoplast microtubules and where pre-prophase bands intersect the edges of cells. It is suggested that the complexes participate in the development of cortical arrays of microtubules. The observations are combined with others, made on pre-prophase bands and on the substructure of cortical arrays lying against the faces of cells, to develop an hypothesis on the development of cortical microtubules, summarised below: Microtubules are nucleated along the edges of cells, at first growing in unspecified orientations and then becoming bridged to the plasma membrane. Parallelism of microtubules in the arrays arises by inter-tubule cross-bridging. Lengths of microtubule are released from, or break off, the nucleating centres and are moved out onto the face of the cell by intertubule and tubule-membrane sliding, thus accounting for the presence there of short tubules with randomly placed terminations. The nucleating zones along cell edges might have vectorial properties, and thus be able to control the orientation of the microtubules on the different faces of the cell. Also, localised activation could generate localised arrays, especially pre-prophase bands in specified sites and planes. Two possible reasons for the spatial restriction of nucleation to cell edges are considered. One is that the geometry of an edge is itself important; the other is that along most cell edges there is a persistent specialised zone, inherited at cytokinesis by the daughter cells when the cell plate bisects the former pre-prophase-band zone.

The length and disposition of cortical microtubules in plant cells fixed in glutaraldehyde-Osmium tetroxide.

Serial sectioning has been used to show that the majority of circumferential microtubules lying in the cortex of root tip cells are much shorter than the cell circumference. The significance of this observation is briefly discussed.