Membrane fluidity determines sensitivity of filamentous fungi to chitosan.

The antifungal mode of action of chitosan has been studied for the last 30 years, but is still little understood. We have found that the plasma membrane forms a barrier to chitosan in chitosan-resistant but not chitosan-sensitive fungi. The plasma membranes of chitosan-sensitive fungi were shown to have more polyunsaturated fatty acids than chitosan-resistant fungi, suggesting that their permeabilization by chitosan may be dependent on membrane fluidity. A fatty acid desaturase mutant of Neurospora crassa with reduced plasma membrane fluidity exhibited increased resistance to chitosan. Steady-state fluorescence anisotropy measurements on artificial membranes showed that chitosan binds to negatively charged phospholipids that alter plasma membrane fluidity and induces membrane permeabilization, which was greatest in membranes containing more polyunsaturated lipids. Phylogenetic analysis of fungi with known sensitivity to chitosan suggests that chitosan resistance may have evolved in nematophagous and entomopathogenic fungi, which naturally encounter chitosan during infection of arthropods and nematodes. Our findings provide a method to predict the sensitivity of a fungus to chitosan based on its plasma membrane composition, and suggests a new strategy for antifungal therapy, which involves treatments that increase plasma membrane fluidity to make fungi more sensitive to fungicides such as chitosan.

Chitosan permeabilizes the plasma membrane and kills cells of Neurospora crassa in an energy dependent manner.

Chitosan has been reported to inhibit spore germination and mycelial growth in plant pathogens, but its mode of antifungal action is poorly understood. Following chitosan treatment, we characterized plasma membrane permeabilization, and cell death and lysis in the experimental model, Neurospora crassa. Rhodamine-labeled chitosan was used to show that chitosan is internalized by fungal cells. Cell viability stains and the calcium reporter, aequorin, were used to monitor plasma membrane permeabilization and cell death. Chitosan permeabilization of the fungal plasma membrane and its uptake into fungal cells was found to be energy dependent but not to involve endocytosis. Different cell types (conidia, germ tubes and vegetative hyphae) exhibited differential sensitivity to chitosan with ungerminated conidia being the most sensitive.

Algorithms for the automated analysis of cellular dynamics within living fungal colonies.

We present robust and efficient algorithms to automate the measurement of nuclear movement and germ tube extension rates in living fungal networks. The aim is to facilitate the understanding of the dynamics and regulation of nuclear migration in growing fungal colonies. The proposed methodology combines a cascade correlation filter to identify nuclear centers from which 2D nuclear velocities are determined and a level set algorithm for centerline extraction to monitor spore (conidial) germling growth. We show how the proposed cascaded filter improves spatial resolution in the presence of noise and is robust when fluorescently labeled nuclei with different intensities are in close proximity to each other. The performance of the filter is evaluated by simulation in comparison to the well known Rayleigh and Sparrow criteria, and experimental evidence is given from clusters of nuclei and nuclei undergoing mitotic division. The capabilities developed have enabled the robust and objective analysis of 10's of Gigabytes of image data that is being exploited by biological scientists.

Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins.

The genetic transformation of the higher plant Nicotiana plumbaginifolia to express the protein apoaequorin has recently been used as a method to measure cytosolic free calcium ([Ca2+]i) changes within intact living plants (Knight, M. R., A. K. Campbell, S. M. Smith, and A. J. Trewavas. 1991. Nature (Lond.). 352:524-526; Knight, M. R., S. M. Smith, and A. J. Trewavas. 1992. Proc. Natl. Acad. Sci. USA. 89:4967-4971). After treatment with the luminophore coelenterazine the calcium-activated photoprotein aequorin is formed within the cytosol of the cells of the transformed plants. Aequorin emits blue light in a dose-dependent manner upon binding free calcium (Ca2+). Thus the quantification of light emission from coelenterazine-treated transgenic plant cells provides a direct measurement of [Ca2+]i. In this paper, by using a highly sensitive photon-counting camera connected to a light microscope, we have for the first time imaged changes in [Ca2+]i in response to cold-shock, touch and wounding in different tissues of transgenic Nicotiana plants. Using this approach we have been able to observe tissue-specific [Ca2+]i responses. We also demonstrate how this method can be tailored by the use of different coelenterazine analogues which endow the resultant aequorin (termed semi-synthetic recombinant aeqorin) with different properties. By using h-coelenterazine, which renders the recombinant aequorin reporter more sensitive to Ca2+, we have been able to image relatively small changes in [Ca2+]i in response to touch and wounding: changes not detectable when standard coelenterazine is used. Reconstitution of recombinant aequorin with another coelenterazine analogue (e-coelenterazine) produces a semi-synthetic recombinant aequorin with a bimodal spectrum of luminescence emission. The ratio of luminescence at two wavelengths (421 and 477 nm) provides a simpler method for quantification of [Ca2+]i in vivo than was previously available. This approach has the benefit that no information is needed on the amount of expression, reconstitution or consumption of aequorin which is normally required for calibration with aequorin.

Role of Calcium in Signal Transduction of Commelina Guard Cells.

The role of cytosolic Ca2+ in signal transduction in stomatal guard cells of Commelina communis was investigated using fluorescence ratio imaging and photometry. By changing extracellular K+, extracellular Ca2+, or treatment with Br-A23187, substantive increases in cytosolic Ca2+ to over 1 micromolar accompanied stomatal closure. The increase in Ca2+ was highest in the cytoplasm around the vacuole and the nucleus. Similar increases were observed when the cells were pretreated with ethyleneglycol-bis-(o-aminoethyl)tetraacetic acid or the channel blocker La3+, together with the closing stimuli. This suggests that a second messenger system operates between the plasma membrane and Ca2+-sequestering organelle(s). The endogenous growth regulator abscisic acid elevated cytosolic Ca2+ levels in a minority of cells investigated, even though stomatal closure always occurred. Ca2+-dependent and Ca2+-independent transduction pathways linking abscisic acid perception to stomatal closure are thus indicated.

Calcium Channel Activity during Pollen Tube Growth and Reorientation.

We have shown previously that the inhibition of pollen tube growth and its subsequent reorientation in Agapanthus umbellatus are preceded by an increase in cytosolic free calcium ([Ca2+]c), suggesting a role for Ca2+ in signaling these processes. In this study, a novel procedure was used to measure Ca2+ channel activity in living pollen tubes subjected to various growth reorienting treatments (electrical fields and ionophoretic microinjection). The method involves adding extracellular Mn2+ to quench the fluorescence of intracellular Indo-1 at its ca2+-insensitive wavelength (isosbestic point). The spatial and temporal kinetics of Ca2+ channel activity correlated well with measurements of [Ca2+]c dynamics obtained by fluorescence ratio imaging of Indo-1. Tip-focused gradients in Ca2+ channel activity and [Ca2+]c were observed and quantified in growing pollen tubes and in swollen pollen tubes before reoriented growth. In nongrowing pollen tubes, Ca2+ channel activity was very low and [Ca2+]c gradients were absent. Measurements of membrane potential indicated that the growth reorienting treatments induced a depolarization of the plasma membrane, suggesting that voltage-gated Ca2+ channels might be activated.

Model of hyphal tip growth involving microtubule-based transport.

We propose a simple model for mass transport within a fungal hypha and its subsequent growth. Inspired by the role of microtubule-transported vesicles, we embody the internal dynamics of mass inside a hypha with mutually excluding particles progressing stochastically along a growing one-dimensional lattice. The connection between long-range transport of materials for growth and the resulting extension of the hyphal tip has not previously been addressed in the modeling literature to our knowledge. We derive and analyze mean-field equations for the model and present a phase diagram of its steady-state behavior, which we compare to simulations. We discuss our results in the context of the filamentous fungus Neurospora crassa.

Time-Multiplexed Laguerre-Gaussian holographic optical tweezers for biological applications.

A ferroelectric liquid crystal spatial light modulator is used to generate up to 24 independently controllable traps in a holographic optical tweezers system using time-multiplexed Fresnel zone plates. For use in biological applications, helical zone plates are used to generate Laguerre-Gaussian laser modes. The high speed switching of the ferroelectric device together with recent advances in computer technology enable fast, smooth movement of traps that can be independently controlled in real time. This is demonstrated by the trapping and manipulation of yeast cells and fungal spores.

Calcium waves and dynamics visualized by confocal microscopy in Xenopus oocytes expressing cloned TRH receptors.

Laser scanning confocal microscopy was used to analyse changes in free cytosolic calcium ([Ca2+]i) in Xenopus laevis oocytes expressing the cloned rat TRH receptor in response to TRH. In oocytes expressing TRH receptors, TRH invariably evoked a dose-dependent, biphasic calcium response. This response consisted of an initial transient planar wave of calcium propagating just below the surface of the membrane followed by a slower, secondary calcium phase. The TRH antagonist, chlordiazepoxide, markedly inhibited this calcium wave. The origins of calcium involved in this biphasic response were investigated using a variety of intra- and extra-cellular calcium antagonists. The intracellular calcium antagonists thapsigargin and TMB-8 reduced the initial and to a lesser extent the secondary phase of the planar calcium wave. In contrast, EGTA and the calcium channel blocker nifedipine produced a profound inhibition of the secondary phase while the initial phase was only slightly reduced. These results indicate that the release of intracellular calcium is predominantly responsible for the initial phase of the calcium wave while the influx of extracellular calcium is mainly involved in the secondary phase. Qualitative changes in the patterns of calcium release induced by TRH were observed following pretreatment with intracellular calcium antagonists. Following pretreatment with these compounds, TRH induced spiral or regenerative calcium waves. Addition of EGTA to the extracellular medium did not alter these responses confirming the importance of intracellular calcium in the generation of these spiral calcium waves. This study demonstrates the nature and multiplicity of regulating mechanisms of [Ca2+]i following activation of TRH receptors expressed in Xenopus oocytes.

Low-temperature scanning electron microscopy in biology.

A review of low-temperature scanning electron microscopy (LTSEM) with regard to preparation protocols, specimen preservation, experimental approaches, and high-resolution studies, is provided. Preparative procedures are described and recent developments in methodologies highlighted. It is now well established that LTSEM, for most biological specimens, provides superior specimen preservation than does ambient-temperature SEM. This is because frozen-hydrated samples retain most or all of their water, are rapidly immobilized and stabilized by cryofixation, and are not exposed to chemical modification or solvent extraction. Nevertheless, artefacts in LTSEM are common and most arise because frozen-hydrated specimens contain water. LTSEM can be used as a powerful experimental tool. Advantages of employing LTSEM for this purpose and ways in which it can be used for novel experimentation are discussed. The most exciting development in recent years has been high-resolution LTSEM. The advantages, problems and requirements for this approach are defined.

Effects of hydration on the surface morphology of urediospores.

Changes in the surface morphology of mature urediospores of Uromyces viciae-fabae have been studied with respect to different preparative procedures for the scanning electron microscope. Specimens were examined in the critical point-dried, freeze-dried and frozen-hydrated states. Dried urediospores were significantly different in appearance from those which were hydrated. Shrinkage probably accounted for some of the surface features of dried spores, particularly amongst those which had been critical point-dried. No significant effects on spore surface texture resulted from chemical immersion fixation, chemical vapour fixation, dehydration or the mode of sputter-coating. It is suggested that the presence or absence of unfreezable water, involved in maintaining the structural stability of polymers which provide the specimen surface with its characteristic morphology, may have an important influence on the gross texture of cell surfaces as viewed with the scanning electron microscope.

Expression of recombinant aequorin as an intracellular calcium reporter in the phytopathogenic fungus Phyllosticta ampelicida.

Conidia of Phyllosticta ampelicida germinate only after they have made contact with a substratum. Previous work has shown that external free calcium must be available to the spore for germination to be initiated. Transgenic strains of P. ampelicida expressing apo-aequorin, a calcium-sensitive luminescent protein, were developed to monitor cytoplasmic free Ca(2+) ([Ca(2+)]c). Transformants were verified by PCR and Southern hybridization. Apo-aequorin production was quantified for each of 21 transformants. The transformant that emitted the most light per unit of protein was found to contain 0.59 mg apo-aequorin/g total protein. To ascertain the feasibility of aequorin-based [Ca(2+)]c quantification, [Ca(2+)]c changes were measured in mycelia during various physiologically perturbing treatments: exposure to high concentrations of external Ca(2+), hypoosmotic shock, and mechanical perturbation. This is the first report of a plant pathogenic fungus for which aequorin-based Ca(2+) measurement protocols have been developed.

The promoter of a basic PR1-like gene, AtPRB1, from Arabidopsis establishes an organ-specific expression pattern and responsiveness to ethylene and methyl jasmonate.

Antimicrobial proteins are a key feature underlying the deployment of both pre-formed and inducible defence responses. Probably the most well characterised class are the pathogenesis-related (PR) proteins, which are found in both basic and acidic isoforms. Here we describe the isolation and characterisation of a gene, designated AtPRB1, encoding a basic PR1-like protein from Arabidopsis. This protein showed high amino acid sequence identity with basic and acidic PR1 proteins from other plant species, for example PRB1 from Nicotiana tabacum and PR1 from Brassica napus, at 64% and 78% identity respectively. A genomic DNA fragment containing 2345 bp upstream from the putative transcriptional start site was fused to the gene encoding the luciferase (LUC) gene from Photinus pyralis in order to test for promoter activity. The resulting construct was transformed into Arabidopsis accession Col-0 and analysis of LUC activity, using an ultra-low-light imaging camera system, revealed that the AtPRB1 promoter established an exquisite organ-specific expression pattern. LUC activity was observed in flowers, stems and roots but not in leaf tissue. Superimposed upon this organ-specific expression pattern was responsiveness, in root tissue, to ethylene and methyl jasmonate (MeJA), important cues during the establishment of plant disease resistance. In contrast, AtPRB1::LUC gene expression was repressed in response to salicylic acid treatment. Analysis of a limited series of AtPRB1 5'-promoter deletion mutants, identified a number of promoter regions important for both the establishment of organ-specific expression and responsiveness to ethylene and MeJA. While AtPRB1 gene expression was not induced in response to an avirulent isolate of Peronospora parasitica in leaf tissue, this gene may contribute to horizontal resistance in other tissues and/or to MeJA- and ethylene-dependent defence responses engaged against necrotrophic pathogens in root tissue. It is anticipated that transgenic plants containing AtPRB1-based promoter::reporter constructs will provide useful tools for the future dissection of the cognate signalling networks regulating the expression of this gene.

Visualisation and measurement of the calcium message in guard cells.

We have applied several novel technologies to investigate the role of cytosolic free calcium [Ca2+]i in signal transduction in guard cells of Commelina communis L. Fluorescence ratio imaging and photometry together with the fluorescent Ca2+ indicator Indo-1 were used to directly visualise and measure dynamic spatial and temporal changes in [Ca2+]i in response to various exogenous stimuli. More subtle manipulation of the Ca2+ signal transduction pathway was achieved through the use of photoactivateable, caged Ca2+ and caged inositol-1,4,5-triphosphate (InsP3) released directly into the cytoplasm of the guard cell after microinjection. In these experiments, changes in [Ca2+]i were simultaneously monitored with the fluorescent Ca2+ indicator, Fluo-3. Resting levels of [Ca2+]i (100-200 nM) increased in response to elevated [Ca2+]e, lowering [K+]e, application of the ionophore A-23187 or cytosolic release of either Ca2+ or InsP3 from their caged forms. Stomatal closure was triggered if [Ca2+]i increased above a threshold of about 600 nM. Abscisic acid (ABA) had little effect on [Ca2+]i in the majority of cells studied, being elevated in only a minority of cells investigated. However, stomatal closure occurred in all cases after ABA application. This suggests that ABA acts through both Ca(2+)-independent and Ca(2+)-dependent pathways. The imaging data revealed a substantial heterogeneity in [Ca2+]i within the guard cell. Cytoplasmic regions, particularly near the nucleus, often showed marked elevations and sometimes oscillations. The origin and kinetics of the Ca2+ fluxes leading to the dynamic spatial patterns is discussed along with several new approaches directed towards identification of the source of the Ca2+. These methods include optical sectioning and 3-D reconstruction of both the endomembrane system and [Ca2+]i in living guard cells using confocal microscopy. Overall, our data is consistent with multiple sources for [Ca2+]i, including uptake across the plasma membrane and InsP3- or Ca(2+)-induced Ca2+ release from internal stores.

FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells.

FM-dyes are widely used to study endocytosis, vesicle trafficking and organelle organization in living eukaryotic cells. The increasing use of FM-dyes in plant cells has provoked much debate with regard to their suitability as endocytosis markers, which organelles they stain and the precise pathways they follow through the vesicle trafficking network. A primary aim of this article is to assess critically the current status of this debate in plant cells. For this purpose, background information on the important characteristics of the FM-dyes, and of optimal dye concentrations, conditions of dye storage, and staining and imaging protocols, are provided. Particular emphasis is placed on using the FM-dyes in double labelling experiments to identity specific organelles. In this way, staining of the Golgi with FM4-64 has been demonstrated for the first time.

Water droplets and ice deposits in leaf intercellular spaces: redistribution of water during cryofixation for scanning electron microscopy.

An experimental study is described of the formation of extracellular deposits on the surfaces of cells in freeze-fractured, frozen-hydrated primary leaves of Phaseolus vulgaris examined by low-temperature scanning electron microscopy. The deposits, observed under a range of experimental conditions, consisted of (a) droplets with diameters of 1.5 to 3.0 µm, (b) droplets with diameters of 10 to 30 µm, (c) crystals with diameters of 1.0 to 6.0 µm, and (d) granules with diameters up to 0.15 µm. The types of deposit were influenced by specimen cooling rate, and their distribution was influenced by the direction of the thermal gradient during cooling. All deposits were predominantly water ice. The quantities of deposited water (up to 4.0% of the leaf water content) increased as the cooling rate was reduced. It is concluded that the ice deposits were primarily artefacts of cryofixation and do not represent the location of water in vivo, as recently suggested. We propose that the deposits arose in four main ways: (1) displacement of water from underlying cells by a pressure wave resulting from the volume increase of intracellular water as it freezes, (2) evaporation of water from warmer cells and its condensation onto colder cells, (3) withdrawal of water from underlying cells by extracellular ice crystallization, (4) condensation of pre-existing water vapour in the intercellular spaces onto cells. The significance of the findings is discussed in relation to the use of lowtemperature scanning electron microscopy in studies of plant morphology and for localizing water and soluble ions within plant cells and tissues.

Dynamic distribution of BIMG(PP1) in living hyphae of Aspergillus indicates a novel role in septum formation.

Mutation of bimG, the major protein phosphatase 1 gene in Aspergillus nidulans, causes multiple cell cycle and hyphal growth defects that are associated with overphosphorylation of subcellular components. We have used functional translational fusions with the green fluorescent protein (GFP) to show that BIMG has at least four discrete locations within growing hyphae. Three of these locations, the hyphal tip, the spindle pole body and the nucleus, correlate with previously known requirements for bimG(PP1) in mitosis and hyphal growth and are highly dynamic. BIMG-GFP in the hyphal tip seemed to be associated with the plasma membrane and formed a collar of fluorescence within the apical dome. The distribution of nuclear BIMG-GFP varied depending on nutritional conditions; on poor medium, it concentrated more in the nucleolus than in the nucleoplasm, whereas on rich medium, it was more evenly distributed between the two nuclear regions. The association of BIMG-GFP with developing septa was transient, and we present evidence that BIMG phosphatase plays a direct role in septum formation, distinct from its role in mitosis. We conclude that, by being physically present at several sites, the BIMG phosphatase has roles in multiple cellular processes.

Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae.

Confocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were treated with FM4-64, FM1-43 or TMA-DPH, three of the most commonly used membrane-selective dyes reported as markers of endocytosis. All three dyes were rapidly internalized within hyphae. FM4-64 was found best for imaging the dynamic changes in size, morphology and position of the apical vesicle cluster within growing hyphal tips because of its staining pattern, greater photostability and low cytotoxicity. FM4-64 was taken up into both the apical and subapical compartments of living hyphae in a time-dependent manner. The pattern of stain distribution was broadly similar in a range of fungal species tested (Aspergillus nidulans, Botrytis cinerea, Magnaporthe grisea, Neurospora crassa, Phycomyces blakesleeanus, Puccinia graminis, Rhizoctonia solani, Sclerotinia sclerotiorum and Trichoderma viride). With time, FM4-64 was internalized from the plasma membrane appearing in structures corresponding to putative endosomes, the apical vesicle cluster, the vacuolar membrane and mitochondria. These observations are consistent with dye internalization by endocytosis. A speculative model of the vesicle trafficking network within growing hyphae is presented.

Pronounced cytoplasmic pH gradients are not required for tip growth in plant and fungal cells.

The existence of pronounced cytoplasmic pH gradients within the apices of tip-growing cells, and the role of cytoplasmic pH in regulating tip growth, were investigated in three different cell types: vegetative hyphae of Neurospora crassa; pollen tubes of Agapanthus umbellatus; and rhizoids of Dryopteris affinis gametophytes. Examination of cytoplasmic pH in growing cells was performed by simultaneous, dual emission confocal ratio imaging of the pH-sensitive probe carboxy SNARF-1. Considerable attention was paid to the fine tuning of dye loading and imaging parameters to minimise cellular perturbation and assess the extent of dye partitioning into organelles. With optimal conditions, cytoplasmic pH was measured routinely with a precision of between +/-0.03 and +/-0.06 of a pH unit and a spatial resolution of 2.3 microm2. Based on in vitro calibration, estimated values of mean cytoplasmic pH for cells loaded with dye-ester were between 7.15 and 7.25 for the three cell types. After pressure injecting Neurospora hyphae with dextran-conjugated dye, however, the mean cytoplasmic pH was estimated to be 7.57. Dextran dyes are believed to give a better estimate of cytoplasmic pH because of their superior localisation and retention within the cytosol. No significant cytoplasmic pH gradient (delta pH of >0.1 unit) was observed within the apical 50 microm in growing cells of any of the three cell types. Acidification or alkalinisation of the cytoplasm in Neurospora hyphae, using a cell permeant weak acid (propionic acid at pH 7.0) or weak base (trimethylamine at pH 8.0), slowed down but did not abolish growth. However, similar manipulation of the cytoplasmic pH of Agapanthus pollen tubes and Dryopteris rhizoids completely inhibited growth. Modification of external pH affected the growth pattern of all cell types. In hyphae and pollen tubes, changes in external pH were found to have a small transient effect on cytoplasmic pH but the cells rapidly readjusted towards their original pH. Our results suggest that pronounced longitudinal gradients in cytoplasmic pH are not essential for the regulation of tip growth.

Calcium measurement in living filamentous fungi expressing codon-optimized aequorin.

Calcium signalling is little understood in filamentous fungi largely because easy and routine methods for calcium measurement in living hyphae have previously been unavailable. We have developed the recombinant aequorin method for this purpose. High levels of aequorin expression were obtained in Neurospora crassa, Aspergillus niger and Aspergillus awamori by codon optimization of the aequorin gene. Three external stimuli (mechanical perturbation, hypo-osmotic shock and high external calcium) were found transiently to increase [Ca(2+)](c). Each of the calcium signatures associated with these physico-chemical treatments was unique, suggesting the involvement of three distinct calcium-mediated signal transduction pathways. The fungal calcium channel blocker KP4 inhibited the [Ca(2+)](c) responses to hypo-osmotic shock and high external calcium, but not to mechanical perturbation. The divalent cation chelator BAPTA inhibited [Ca(2+)](c) responses to mechanical perturbation and hypo-osmotic shock. The calcium agonists A23187 and cyclopiazonic acid increased [Ca(2+)](c) levels.