In the field, pathogens exist in a diverse and dynamic ecological environment with other organisms (Blakeman, 1993; Preece & Dickinson, 1971). We tend to assume that these other organisms are ‘benign’ or perhaps in competition with the pathogen to occupy a particular niche. However, bacteria are often observed to be in close association with pathogens, as witnessed by the difficulty of eliminating them during isolation of pathogens involved in plant diseases. We now have clear evidence (Dewey et al., pp. 489–497 in this issue) that they are sometimes neither benign, nor in competition, but actually helping the pathogen successfully infect its host even when the bacteria themselves are not pathogens of that host.

CONTENTS

Summary 389

I. INTRODUCTION 389

II. 1. The SNARE hypothesis 393

2. SNAREs and associates 395

(a) NSF 395

(b) SNAP 395

(c) SNARE 395

(d) Rab and Rab-associated proteins 397

(e) Other interacting proteins 398

(f) Clostridial neurotoxins 399

3. New perspectives on SNARE function 400

(a) Revisiting the SNARE hypothesis 400

(b) Roles for SNARE proteins 401

III. VESICLE TRAFFICKING IN PLANTS 402

1. Identifying secretory proteins in plants 402

2. Placing syntaxins in order 403

3. Functional analysis of plant SNAREs in vesicle trafficking 404

4. Small GTPases 405

5. Searching for a vesicle–fusion complex 405

IV. REGULATION OF VESICLE TRAFFICKING IN PLANT CELLS 406

1. Hormonal control of secretion 406

(a) Auxin-evoked secretion and cell growth 406

(b) Gibberellic acid and secretion by barley aleurone 407

(c) Abscisic acid control of stomatal aperture 407

2. Coupling secretion to cytosolic-free Ca+ concentration 408

3. Stress-evoked secretion 409

(a) Mechanical stress 409

(b) Wounding and pathogen attack 409

V. CONCLUSION 410

Acknowledgements 411

References 411

Eukaryotic cells share a set of secretory pathways for the flux of membrane and protein material. In 1993, ideas about the functioning of three major proteins of the neurosecretory complex were consolidated in the SNARE hypothesis, which proposed that the interaction of these proteins provides both the specificity for vesicle targeting and the molecular machinery for fusion between vesicle and target membranes. Subsequetly, the organization, molecular mechanics and control of vesicle trafficking have become topics of intense research, and the hypothesis has evolved to accommodate new discoveries from the analysis of secretion in yeast and mammals. It is likely to be challenged again as more information comes to light about secretory processes in plants. New tools for measuring and manipulating vesicle traffic and secretion are now being used, drawing on in vivo fluorescence and capacitance recording as well as genetic engineering. These new technologies have already begun to yield details wholly unexpected from past studies. Here we focus on recent findings relating to the mechanisms of vesicle trafficking and the background to these developments, highlighting both current understanding of the molecular events of secretion and the gaps therein, as well as discussing emerging themes from work with plants.

Cryptogamous plants in the forest communities of Bialowieza National Park. Functional groups analysis and general synthesis. Ed. by J. B. FALINSKI and W. MULENKO. 30×21 cm. Pp. 224 with 4 plates and numerous text-figures. Phytocoenosis (N. S.) 8, Archivum Geobotanicum 6. 1996. P/b.

Cryptogamous plants in the forest communities of Bialowieza National Park. Ecological atlas of seminal and cryptogamous plants. Ed. by J. B. FALINSKI and W. MULENKO. 30×21 cm. Pp. 522 with 2000 maps. Phytocoenosis (N. S.) 9, Supplementum Cartographicae Geobotanicae 7. 1997. H/b.

Deciduous woody pioneer species (Juniperus communis, Populus tremula, Salix sp. div.) in the secondary succession and regeneration. By J. B. FALINSKI. 30×21 cm. Pp. 156 with 8 plates, 2 end-paper charts and numerous text-figures. Phytocoenosis (N. S.) 10, Supplementum Cartographicae Geobotanicae 8. 1998. P/b.

Synanthropization of plant cover in new Polish research. Ed. by J. B. FALINSKI, W. ADAMOWSKI and B. JACKOWIAK. 30×21 cm. Pp. 279 with 2 plates, 2 end-paper maps and numerous text-figures. Phytocoenosis (N. S.) 10, Supplementum Cartographicae Geobotanicae 9. 1998. P/b.

The relative ozone sensitivities of 25 German native herbaceous plant species, representative of arable field margins or disturbed habitats, were examined over three consecutive growing seasons. Plants were grown from seed, potted into natural soils and exposed in open-top chambers for the entire season to different ozone-exposure regimes covering a range of concentrations from <5 to 48 ppb (seasonal 8 h daily mean). The assessment of ozone effects was carried out by recording the first day of visible symptom appearance and the percentage of injured leaves at the end of vegetative growth. Species exhibited contrasting patterns of symptom expression under ozone stress, with either ozone-specific symptoms or ozone-enhanced foliar pigmentation and senescence. Classifications of species according to their ozone susceptibility varied depending on whether measurement was of the total extent of visible injury, ozone threshold doses for the incidence of symptoms, or modelled exposure–response relationships. The most sensitive species exhibiting ozone-specific symptoms were Cirsium arvense and Sonchus asper, which responded to accumulated ozone exposures <1500 ppb.h (AOT40). For these and three other species, an AOT40 peak of a single day was found to be responsible for the incidence of ozone-specific symptoms, i.e. injury occurred rapidly within a few days of the day with the highest AOT40, while other species responded only to longer-term ozone exposures. The relative ozone sensitivity of the species was calculated by combining the different sensitivity criteria, and possible systematic trends (taxonomic or evolutionary features) are pointed out. The results suggest it may be possible to use a particular group of native herbaceous plant species with contrasting patterns of ozone sensitivity as a biomonitoring system in the field. This allows plant responses to be related either to peak values or to prolonged ozone exposure, making it possible to distinguish between short- and long-term effects of ozone.

Physiological responses of two provenances of European beech (Fagus sylvatica) were studied in seedlings grown at two [CO2] in combination with four temperature treatments. For the local Danish provenance, the average effect of elevated [CO2] during growth was to increase light-saturated net photosynthesis (An) and instantaneous water-use efficiency or transpiration efficiency (ITE). These increases were strongly related to the temperature treatment. Stomatal conductance (gs) was reduced in seedlings in high [CO2], but there was no statistically significant effect of temperature treatment. Stomatal conductance was 13–26% lower at elevated [CO2] and ITE was 89–156% higher, depending on growth temperature. The effects of [CO2] on An were considerably larger than those shown for many other woody species, but similar to those in other studies on European beech. The absolute value of An for a Romanian provenance of beech was 5–18% lower than in the Danish provenance at low [CO2] and 14–26% lower at high [CO2]. There was no statistically significant interaction between the provenances and [CO2], or between provenance and temperature. A model of the response of An to [CO2] at different temperatures gave predictions close to the measured results, except at the lowest temperature treatment where the model over-predicted the effect of elevated [CO2]. This and measurements of An made at a common, low [CO2] indicated a down-regulation of photosynthesis in the lowest temperature treatment at high [CO2]. Root plus soil respiration on a whole-tree basis (Rtr) was increased by elevated [CO2] at all but the lowest temperature, but no effect was seen of [CO2] on root respiration per unit root d. wt. Mean Rtr on any given date was significantly correlated with An, except at the lowest temperature treatment. It is hypothesized that low temperature limited the ability of the roots to use photosynthates resulting in a feedback inhibition of An when elevated [CO2] was combined with low temperature.

To predict changes in the range and density of plant species as a consequence of elevated atmospheric CO2, it is essential to characterize the effect of elevated CO2 on key components of plant life history stages such as rate of establishment and maturation of individuals, reproductive output and offspring fitness. We measured vegetative response, phenology, reproductive output and seed quality of four annual C3 plant species, grown from seed to senescence under ambient and elevated (ambient+200 ppm) CO2 and ambient and reduced (33% shade) light. Few previous studies have included all stages of a plant's life as well as characteristics of the next generation. Elevated CO2 had no effect on the vegetative attributes of Cardamine hirsuta, Spergula arvensis or Poa annua whereas Senecio vulgaris produced longer leaves and greater biomass. Both Senecio and Poa had faster maturation times. The vegetative response of Senecio was not translated into increased seed output, although seed mass and carbon[ratio ]nitrogen ratios were significantly increased. By contrast, Poa showed no vegetative response to elevated CO2, but had significantly increased seed production. Thus we found no evidence for a simple translation from vegetative response to elevated CO2 into reproductive response. There was also no consistent light-mediated response to elevated CO2 among the four species. However, the effect of reduced light (33% shade) on vegetative and reproductive output was consistent across the four species and significantly stronger than the effect of elevated CO2. On the basis of this glasshouse study, we predict that Poa would be most likely of the four species to show significant increases in population size and migration potential, as a result of increased reproductive output, under elevated atmospheric CO2. However, this response may be relatively small compared with variation in growth and reproduction as a result of environmental heterogeneity in resources such as light.

The growth of the marine flagellate Isochrysis galbana was followed in batch cultures at four concentrations of dissolved inorganic carbon (DIC), from C- and N-replete lag phase into C- and/or N-deplete stationary phase. Organic buffers were omitted from the growth medium, and culture pH was maintained at 8.30±0.05 by the addition of acid or alkali. The responses of the flagellate to N stress included an increase in the C[ratio ]N ratio, and decreases in the ratios of glutamine (Gln)[ratio ]glutamate (Glu) and Chl a[ratio ]C, and the cell Chl a quota. Conversely, the responses to C stress included a decrease in the C[ratio ]N ratio, and increases in the ratios of Gln[ratio ]Glu and Chl a[ratio ]C, and the cell Chl a quota. The relationship between carbon-specific growth rate (C-μ), and the concentration of extracellular DIC, [DIC]ext, exhibited Michaelis–Menten type kinetics with a half saturation constant, KG(DIC), of 81 μM. Comparative studies of the diatom Phaeodactylum tricornutum showed similar results, although the value of KG(DIC) was lower at 30 μM.

This paper considers the extent to which differences in pollen tube growth rates can provide prezygotic reproductive isolation between Mimulus nasutus and its presumed progenitor, Mimulus guttatus. Mimulus nasutus is partially cleistogamous, but its larger chasmogamous flowers offer appreciable opportunity for outcrossing. Mimulus nasutus was found to have smaller pollen grains and shorter styles than M. guttatus. No differences were observed in pollen grain germination on conspecific and heterospecific stigmas. However, pollen tube growth rates of M. nasutus were found to be much slower than those of M. guttatus in the styles of that species. Consequently, any M. nasutus pollen transferred to an M. guttatus stigma was found to be competitively disadvantaged in an M. gutattus style. By contrast, no difference in pollen tube growth rate was detected between the species when growing in M. nasutus styles, possibly because M. nasutus styles are unable to support fast pollen tube growth. We tested the prediction from the pollen tube studies that a 50[ratio ]50 mix of M. guttatus and M. nasutus pollen would produce 50% hybrid seeds when M. nasutus was the maternal parent, and near to 0% hybrid seed when M. guttatus was the maternal parent. The results were found to support this prediction. We conclude that pollen–pistil interactions can effect strong reproductive isolation between these species, as M. guttatus pollen tubes have a competitive advantage over those of M. nasutus in an M. guttatus style, but not in an M. nasutus style.

Modern yew (Taxus baccata) is demonstrated to have considerable potential for dendrochronological use. A replicated 303 yr chronology has been formed from Hampton Court Palace yew and cross-matches well with a number of different tree species, including oak. Growth is positively correlated with rainfall but inversely related to late summer temperature. Range limits and present and prehistoric sources of yew are also briefly discussed, highlighting the future potential of yew to become the third major tree species for British dendrochronological study.

In studies with a laboratory isolate of the fungal pathogen Stagonospora (Septoria) nodorum three different isolates of bacteria were closely associated with the fungus. Bacteria were also closely associated with fresh isolates of S. nodorum obtained from artificially and naturally infected field material. Although a range of bacteria was isolated, only one type of bacterium was found to be associated with each isolate of S. nodorum. In co-inoculation studies with pycnidiospores of the fungus on detached leaves, some of the bacterial isolates significantly increased the pathogenicity of the fungus, particularly Xanthomonas maltophilia, Sphingobacterium multivorum, Enterobacter agglomerans and Erwinia amylovora. Evidence is presented indicating that one of the ways that the ‘helper bacteria’ may assist in the establishment of infections is by the production of lipases that were not detected in germinating fungal spores.

The role of cytosolic pH (pHc) in growing germ tubes of the filamentous fungus Magnaporthe grisea was analysed by confocal ratio imaging of the pH-sensitive fluorescent dye 5(6)-carboxyseminaphthorhodafluor-1 (SNARF-1). The cytosol of these cells was successfully loaded with the acetoxymethyl ester of the dye and the pHc was visualized and quantified during conidium germination, germ tube growth and appressorium induction by simultaneous dual-emission confocal ratio imaging. Calibrations of the free acid in vitro and calibrations in vivo produced results indicating a similar dynamic response in the pH range 6.0–8.0 for both methods. The pHc in growing germ tubes was consistently pH 7.2±0.1 during all developmental stages analysed. Only slight changes in pHc (<0.1 pH unit) were found in response to alkaline external pH (pH 8.0). No changes in pHc occurred in response to an acidic extracellular pH (pH 6.0) or to the presence of nutrients. There was no observation of either pronounced gradients or changes in pHc in growing germ tubes accompanying conidium germination, germ tube growth or early appressorium formation.

In vesicular–arbuscular mycorrhizal symbioses, specialized fungal structures (the arbuscules) are formed which are in intimate contact with plant root cortical cells. It is assumed that these arbuscules are the major sites of solute transfer between the plant and fungus, but there have been no studies that definitively show the extent or types of transfer processes that occur in this structure. Phosphate is one of the major nutrients that is acquired by mycorrhizal fungi and transferred to plants. In this study a single Lycopersicon esculentum cDNA was cloned and shown to be identical to LePT1, a previously cloned inorganic-phosphate transporter. Expression studies revealed that LePT1 transcript levels remained constant in mycorrhizal plants, but increased in phosphate-starved, non-mycorrhizal plants. Localization of the LePT1 transcript by in situ hybridization showed that this gene is highly expressed in arbuscule-containing cortical cells in mycorrhizal plants. In non-mycorrhizal plants LePT1 expression was localized to the stele and cortex. The expression studies suggest that this transporter is involved in phosphate nutrition of L. esculentum and its localization in cells that contain arbuscules indicate that it may be the mechanism used by the plant to take up phosphate that is effluxed across the fungal plasma membrane of the arbuscule. Based on our findings and those of others, an integrated model of inorganic phosphate uptake and transfer in mycorrhizal and non-mycorrhizal plants is presented.

Seedlings of the annual grass Vulpia ciliata ssp. ambigua were grown in sterilized sand with a dark septate root fungus, Phialophora graminicola, which had been isolated from a natural population of the grass. Tiller number and shoot, root and total biomass of seedlings grown with P. graminicola were enhanced relative to uninoculated control plants in a growth room and a glasshouse experiment. Root length of seedlings grown with P. graminicola was significantly increased, but no effects of the fungus on root diameter, number of root hairs or specific root length were recorded. Root nitrogen content and shoot, root and total phosphorus contents of seedlings grown with the fungus were enhanced, but shoot nitrogen concentration of these plants was reduced. Shoot biomass and specific root length of inoculated plants were positively associated with the number of P. graminicola colonies re-isolated from roots. These data indicate that P. graminicola acts as a beneficial associate of V. ciliata ssp. ambigua seedlings under controlled conditions.

A systematic survey of the endophytic assemblages of Quercus ilex in central Spain has been performed, with the goal of evaluating the importance of geographical and seasonal factors on these fungal communities. Four sampling sites were selected; one of them was sampled twice, in the spring and the autumn. The collected plant material consisted of bark, twigs and leaves from eight trees per site. Fungal strains were isolated with the use of a surface-sterilization method with sodium hypochlorite. A total of 2921 fungal strains grouped into 149 ‘species’ or morphological types were recovered. The 10 dominant species, with isolation frequencies >1.5%, were Pyrenochaeta sp., Periconiella anamorph of Biscogniauxia mediterranea (De Not.) Kuntze, Pseudonectria sp., Cryptosporiopsis quercina Petrak, Alternaria alternata (Fr:) Keissl., two undetermined coelomycetes, Penicillium funiculosum Thom, Diplodia mutila Fr. apud Mont. and Ascochyta sp. Medians of fungal species per tree were significantly different among the sampled sites. The isolation frequencies of the dominant species, as well as other less frequent species, were significantly dependent on the sampling site. The degree of endophytic infection and the diversity of fungal species were significantly higher in the spring. The frequencies of all dominant species at one of the sites depended significantly on the season, except for C. quercina, Acremonium sclerotigenum (F & V Moreau ex Valenta) Gams. and D. mutila. Cluster analysis of the whole endophytic mycoflora of the sampled trees suggested that the geographical factor affects the endophytic distribution patterns more significantly than the seasonal factor.

Afrothismia winkleri develops fleshy rhizomes, densely covered with small root tubercles, narrowing to filiform roots with age. The exclusively intracellular mycorrhizal fungus has distinct morphologies in different tissues of the plant. In the filiform root the hyphae grow straight and vesicles are borne on short hyphal stalks. The straight hyphae are present in the epidermis of the root tubercles, but change to loosely coiled and swollen hyphae in the rhizome tissue. No penetration from epidermis to root cortex was found. From the rhizome, a separating cell layer permits only one or rarely two hyphal penetrations into the cortex of each root tubercle. The hyphae proceed apically within the root hypodermis in a spiral row of distinctively coiled hyphae, branches of which colonize the inner root cortex. In the inner root cortex the hyphal coils degenerate to amorphous clumps. In older roots the cortex itself also deteriorates, but epidermis, hypodermis, endodermis and central cylinder persist. The mycorrhizal pattern in A. winkleri is interpreted as an elaborate exploitation system whereby the fungus provides carbon and nutrients to the plant and, simultaneously but spatially distinct, its hyphae are used to translocate and store the matter within the plant. Several features indicate that the endophyte is an arbuscular mycorrhizal fungus.

Roots of clonal birches (Betula pendula) were inoculated with the ectomycorrhizal fungi Paxillus involutus (isolates P0 and Mi) and Hebeloma cylindrosporum (strains D1 and D105). These fungi showed different rates of mycorrhiza formation in vitro. Mature mycorrhizas were obtained after only 2–4 d with H. cylindrosporum, whereas 6–8 d were necessary with P. involutus isolate P0, and P. involutus isolate Mi was not able to form mature mycorrhiza during the 10 d of the experiment. Temporal changes in PAL activity and the expression of genes encoding intracellular pathogenesis-related proteins were followed after inoculating birch roots with these fungi. Transient increase of PAL activity, and transient induction of expression of the wound-inducible Bet v1-SC1 gene, were observed in roots challenged with both H. cylindrosporum strains and the P. involutus isolate P0. These changes were found to coincide with hyphal penetration between root cells during Hartig net formation, and were never observed in roots inoculated with the poorly aggressive P. involutus isolate Mi. Examination of mycorrhizal root sections under u.v. light indicated the presence of phenolic compounds in the host cell walls at the vicinity of the Hartig net. These results strongly suggest that hyphal penetration between the root cells triggers a transient defence response which, in turn, could limit Hartig net formation to the outer layer of the root cortex.

The population dynamics of perennial crop plants are influenced by numerous factors, including management practices. Conditions in the field vary from year to year, and matrix population models are useful for evaluating population behaviour in relation to environmental variability. In Missouri, the stand persistence of birdsfoot trefoil (Lotus corniculatus), a perennial legume, is often limited by disease and poor seed production. A stage-based, matrix population model was developed to evaluate the population dynamics of birdsfoot trefoil in relation to clipping treatment. The plant growth stages represented in the model were seeds, seedlings, mature vegetative and reproductive plants. Two phases of population growth were evaluated in clipped and unclipped stands. Establishment-phase populations were characterized by relatively high mortality and low reproduction. Elasticity analysis indicated that growth of these populations was most sensitive to the survival of vegetative plants. Mature vegetative plants and seeds comprised the majority of surviving individuals in clipped and unclipped populations, respectively; however, establishment-phase populations under both management treatments tended toward extinction. Populations in the post-establishment phase of growth were characterized by relatively low mortality and high reproduction. Population growth in this phase of growth was most sensitive to seed production, and most individuals in these populations were at the seed stage.