Oomycete Achlya Bisexualis Research Articles

Biomechanical responses of encysted zoospores of the oomycete Achlya bisexualis to hyperosmotic stress are consistent with an ability to turgor regulate

Zoospores are motile, asexual reproductive propagules that enable oomycete pathogens to locate and infect new host tissue. While motile, they have no cell wall and maintain tonicity with their external media using water expulsion vacuoles. Once they locate host tissue, they encyst and form a cell wall, enabling the generation of turgor pressure that will provide the driving force for germination and invasion of the host. It is not currently known how these spores respond to the osmotic stresses that might arise due to different environments on and around their hosts that have different osmotic strengths. We have made microaspiration (MA) measurements on > 800 encysted zoospores and atomic force microscopy (AFM) measurements on 12 encysted zoospores to determine their mechanical properties and how these change after hyperosmotic stress. Two types of encysted zoospores (Type A and Type B) were produced from the oomycete Achlya bisexualis, that differed in their morphology and response. With a small hyperosmotic stress (using 0.1 and 0.2 M sorbitol to give media osmolality changes of 155.4 and 295.6 mOsmol/kg), Type A zoospores initially became stiffer, with an increase in the Young’s modulus (E) over 30 mins from 0.16 MPa to 0.25 and 0.22 MPa respectively. E then returned to its original value after 120 min. With a greater osmotic stress (using 0.3, 0.4 and 0.5 M sorbitol to give media osmolality changes of 438.2, 587.2 and 787.6 mOsmol/kg) the reverse occurred, with an initial decrease in E over 30 – 60 mins to values of 0.1, 0.08 and 0.09 MPa respectively, before recovery to the original value after 120 min. In 0.5 M sorbitol this recovery was only observed with AFM, but not with MA. Type B zoospores, which may be primary/secondary spores about to release secondary/tertiary spores, or else spores that were damaged during encystment, initially stiffened in response to the lower hyperosmotic stresses with a slight increase in E (from 0.077 to 0.1 MPa after 15 min (with both 0.1 and 0.2 M sorbitol) before recovering to the original value after 60 min. These spores showed no change in response to the higher osmotic stresses. The responses of the Type A spores are consistent with rapid changes in cell wall thickness and a turgor regulation mechanism. Turgor regulation is further supported by microscopic observations of the Type A spores showing protoplast retraction from the cell wall followed by deplasmolysis, coupled with measurements of spore volume. As far as we are aware this is the first demonstration of turgor regulation, not just in encysted zoospores, but in oomycetes in general.

Microfluidic platform for integrated compartmentalization of single zoospores, germination and measurement of protrusive force generated by germ tubes.

This paper describes the design, fabrication and characterisation of a novel monolithic lab-on-a-chip (LOC) platform combining the trapping and germination of individual zoospores of the oomycete Achlya bisexualis with elastomeric micropillar-based protrusive force sensing. The oomycetes are of significant interest due to their pathogenic capabilities, which can have profound ecological and economic impacts. Zoospore encystment and germination via a germ tube play a key role in their pathogenicity. Our platform enables the study of these processes at a single cell level through hydrodynamic trapping of zoospores and their individual compartmentalization via normally closed pneumatic membrane microvalves. Valve geometry was optimized and media exchange characterized during dynamic valve operations to enhance the capture-to-growth ratio. We demonstrate germination of A. bisexualis zoospores on the platform and report three distinct germination patterns. Once germinated, germ tubes grew down growth channels towards single elastomeric micropillars. Tracking of pillar movement allowed for the measurement of microNewton range protrusive forces imparted by the tips of the germ tubes. Results indicate that the forces generated by the germ tubes are smaller than those exerted by mature hyphae. Through the use of parallel traps, channels and pillars on the same device, the platform enables high-throughput screening (HTS) of zoospores and their generation of protrusive force, an essential component of their infective capability. Due to its versatility, it will also allow for the screening of naturally bioactive compounds and the development of new biocontrol strategies for oomycetes, and morphologically similar fungal infections, as an alternative to agrochemicals.

Fabrication of In-Channel High-Aspect Ratio Sensing Pillars for Protrusive Force Measurements on Fungi and Oomycetes

This paper reports the fabrication and application of a Lab-on-a-Chip platform containing single-elastomeric micropillars in channel constrictions, which enable the measurement of protrusive forces exerted by individual fungal hyphae. We show the device design, the fabrication process, and photoresist optimization required to adapt the microfluidic platform to relatively thin hyphae. To demonstrate the applicability of the devices, the oomycete Achlya bisexualis and the fungus Neurospora crassa were cultured on PDMS chips. Devices were combined with confocal imaging to study the interaction of A. bisexualis hyphae with the measurement pillars. The force exerted by individual hyphae of N. crassa was measured and compared with a hyphal growth rate and diameter. The platform provides a new tool to help understand the molecular processes that underlie protrusive growth and this may present new ways to tackle the pathogenic growth of these organisms and thus combat the loss of diversity that they cause. This paper is based on the conference proceedings presented at the 31st IEEE International Conference on Micro Electro Mechanical Systems (MEMS 2018), Belfast. [2018-0090]

A pressure gradient facilitates mass flow in the oomycete Achlya bisexualis.

We have used a single cell pressure probe and observed movement of microinjected oil droplets to investigate mass flow in the oomycete Achlya bisexualis. To facilitate these experiments, split Petri dishes that had media containing different sorbitol concentrations (and hence a different osmotic potential) on each side of the dish were inoculated with a single zoospore. An initial germ tube grew out from this and formed a mycelium that extended over both sides of the Petri dish. Hyphae growing on the 0 M sorbitol side of the dish had a mean turgor ( ± sem) of 0.53 ± 0.03 MPa (n = 13) and on the 0.3 M sorbitol side had a mean turgor ( ± sem) of 0.3 ± 0.027 MPa (n = 9). Oil droplets that had been microinjected into the hyphae moved towards the lower turgor area of the mycelia (i.e. retrograde movement when microinjected into hyphae on the 0 M sorbitol side of the split Petri dish and anterograde movement when microinjected into hyphae on the 0.3 M sorbitol side of the Petri dish). In contrast, the movement of small refractile vesicles occurred in both directions irrespective of the pressure gradient. Experiments with neutral red indicate that the dye is able to move through the mycelia from one side of a split Petri dish to the other, suggesting that there is no compartmentation. This study shows that hyphae that are part of the same mycelia can have different turgor pressures and that this pressure gradient can drive mass flow.

An investigation into plasmolysis in the oomyceteAchlya bisexualisreveals that membrane–wall attachment points are sensitive to peptides containing the sequence RGD and that cell wall deposition can occur despite retraction of the protoplast

The structure and function of membrane-wall attachment sites in walled cells, and how these relate to animal focal adhesions, is an area that is poorly understood. In view of this, we investigated how membrane-wall attachments that form upon plasmolysis, respond to peptides that disrupt animal focal adhesions. The degree of cytoplasmic disruption during plasmolysis was also investigated. Upon hyperosmotic challenge, the protoplast in hyphae of the oomycete Achlya bisexualis typically retracted incompletely due to membrane-wall attachments. The inclusion, in the plasmolysing solution, of peptides containing the sequence RGD disrupted these attachments in a dose-dependent manner. In some hyphae, protoplast retraction stopped temporarily at attachment points - upon resumption of retraction, material was left that traced the outline of the static protoplast. Staining of this material with fluorescence brightener indicated the presence of cellulose, which suggests that wall deposition was able to occur despite plasmolysis. The F-actin cytoskeleton was disrupted during plasmolysis; peripheral F-actin staining was observed, but there was no distinct F-actin cap; staining was more diffuse; and there were fewer plaques compared with nonplasmolysed hyphae. Our data indicate that membrane-wall attachment points are sensitive to RGD-containing peptides and that wall deposition continues despite protoplast retraction and F-actin disruption.

An investigation of the effects of Ca 2+ channel inhibitors on branching and chemotropism in the oomycete Achlya bisexualis: Support for a role for Ca 2+ in apical dominance

In an attempt to better understand branching and chemotropism, we describe the effects of Ca 2+ channel inhibitors on these processes in Achlya bisexualis, using a branch induction technique and whole plate assays. Branching appears to be a two step process with the initial formation of a bump from which a branch emerges. Verapamil increased numbers of branches in whole plate assays and decreased the distance from the first branch to the tip. In induction assays verapamil increased the number of bumps formed, although in some hyphae it inhibited the transition from an initial bump to a branch. When a branch formed it did not affect the time taken to branch. It had no effect on chemotropism. Lanthanum (La 3+) and gadolinium (Gd 3+) also increased branching in whole plate assays but their effect was much less marked and they had no effect on bump/branch number in induction assays. Gd 3+ decreased the time taken to branch. Both La 3+ and Gd 3+ increased chemotropism. These data suggest firstly that the respective inhibitors may affect different parts of the branching process and secondly that Ca 2+ influx through channels may not be a requirement for branching, indeed such movements may suppress branching. This would fit with elevated Ca 2+ at the tip playing a role in apical dominance.

Aphanomyces invadans and Ulcerative Mycosis in Estuarine and Freshwater Fish in Florida

In the spring of 1998, the Florida Fish and Wildlife Research Institute received numerous reports of lesioned or ulcerated fish primarily from the St. Lucie Estuary on the southeast coast of Florida, an area known since the late 1970s for lesions of the ulcerative mycosis (UM) type. From these and archived reports, as well as others received from different areas of Florida, we documented that diseased specimens had randomly distributed skin ulcers (usually reddened or hemorrhagic) with raised irregular margins and, in some cases, deeply penetrating hyphae in the surrounding muscle tissue. Since 1998, 256 fish (comprising 18 species) with ulcerative lesions (from 15 different locations) were confirmed with hyphae in fresh squash preparation or by histological evaluation. Squash preparations revealed nonseptate, sparsely branching, thick-walled hyphae; histological sections revealed mycotic granulomas in the dermis that occasionally penetrated into the skeletal muscle. These pathological characteristics were consistent with UM caused by the oomycete Aphanomyces invadans in Southeast Asia, Japan, Australia, and the United States. For specific identification, six isolates from ulcerated fish were cultured and prepared for molecular characterization using established diagnostic methods. Ribosomal RNA gene sequence analysis identified three isolates as Aphanomyces invadans, one as the oomycete Achlya bisexualis, and two as the ascomycete Phialemonium dimorphosporum. A more extensive survey of 67 ulcerated skin samples from fish collected between 1998 and 2003 was performed using a polymerase chain reaction assay specific for Aphanomyces invadans. Of these, 26 (38.8%) samples from seven fish species and nine collection locations were positive. Confirmation of UM associated with Aphanomyces invadans represents new host records in Florida for the sheepshead Archosargus probatocephalus, striped mullet Mugil cephalus, white mullet Mugil curema, silver perch Bairdiella chrysoura, black drum Pogonias cromis, largemouth bass Micropterus salmoides, and American shad Alosa sapidissima.

A β4 integrin-like protein co-localises with a phosphotyrosine containing protein in the oomycete Achlya bisexualis: Inhibition of tyrosine phosphorylation slows tip growth

We present immunocytochemical data that indicate the presence of, and a close association between β4 integrin-like proteins and proteins containing phosphorylated tyrosine residues in the oomycete Achlya bisexualis. When hyphae were plasmolysed, these proteins were present in wall–membrane attachment sites where there was also F-actin. A combination of immunoblots, ELISA, and a coupled enzyme assay suggest that phosphorylation may occur by both autophosphorylation and through the action of a tyrosine kinase. Tyrphostins, which are inhibitors of tyrosine kinases, abolished the anti-phosphotyrosine staining, inhibited the kinase activity, slowed tip growth and affected the organisation of the actin cytoskeleton, in a dose-dependent manner. By analogy with the integrins and associated kinases of the metazoa we suggest that these proteins could contribute to the process of tip growth by providing a means of bidirectional signaling between the cell wall and the cytoplasm.

Turgor regulation in hyphal organisms

Turgor regulation in two saprophytic hyphal organisms was examined directly with the pressure probe technique. The ascomycete Neurospora crassa, a terrestrial fungi, regulates turgor after hyperosmotic treatments when growing in a minimal medium containing K +, Mg 2+, Ca 2+, Cl −, and sucrose. Turgor recovery by N. crassa after hyperosmotic treatment is concurrent with changes in ion transport: hyperpolarization of the plasma membrane potential and a decline in transmembrane ion conductance. In contrast the oomycete Achlya bisexualis, a freshwater hyphal organism, does not regulate turgor after hyperosmotic treatment, although small transient increases in turgor were occasionally observed. We also monitored turgor in both organisms during hypoosmotic treatment and did not observe a turgor increase, possibly due to turgor regulation. Both hyphal organisms grow with similar morphologies, cellular expansion rates and turgor (0.4–0.7 MPa), yet respond differently to osmotic stress. The results do not support the assumption of a universal mechanism of tip growth driven by cell turgor.

Two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis.

We show that two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis, that have been chemically fixed with a combination of methylglyoxal and formaldehyde and stained with Alexa phalloidin. In approximately one half of the hyphae examined, an F-actin depleted zone within the apical F-actin cap was observed. The remaining hyphae had a continuous apical cap. In live, growing hyphae two types of cytoplasmic organization were observed at the tips, one in which a clear zone was present which may correlate with the F-actin depleted zone, and one where no such clear zone existed which may represent the continuous cap. We suggest that the F-actin depleted zone may be a structural component of the actin network in a subpopulation of oomycete hyphae and may be comparable to similar F-actin depleted zones at the apices of other tip growing cells such as pollen tubes and root hairs. This observation has implications with regard to models of hyphal extension. Hyphae fixed with formaldehyde alone showed continuous apical F-actin caps. Our ability to resolve the F-actin depleted zone likely reflects the cross-linking capabilities of methylglyoxal. The methylglyoxal-formaldehyde combination fixative gave more stained hyphae, brighter staining and more complete staining of F-actin compared to formaldehyde alone.

The pulse of the machine - reevaluating tip-growth methodology.

The pulse of the machine - reevaluating tip-growth methodology.

Heavy metals have different effects on mycelial morphology of Achlya bisexualis as determined by fractal geometry

The morphological response, as measured by changes to mycelial area, radial extension and border fractal dimension, of the oomycete Achlya bisexualis to Cu, Co, Hg, Zn and Cd at concentrations of between 0.05 and 3 mM is described. All of the metals decreased mycelial area and radial extension. Border fractal dimension increased in the presence of Cu, Co and Hg with individual hyphae extending out beyond the mycelial margin. In the presence of 3 mM Hg these hyphae displayed spiral growth. Zn and Cd had no effect on border fractal dimension. We suggest that all of the metals slow growth and that Cu, Co and Hg may also disrupt the relationship between tip growth and branching at the edge of the mycelium.

Heavy metals have different effects on mycelial morphology of Achlya bisexualis as determined by fractal geometry.

The morphological response, as measured by changes to mycelial area, radial extension and border fractal dimension, of the oomycete Achlya bisexualis to Cu, Co, Hg, Zn and Cd at concentrations of between 0.05 and 3 mM is described. All of the metals decreased mycelial area and radial extension. Border fractal dimension increased in the presence of Cu, Co and Hg with individual hyphae extending out beyond the mycelial margin. In the presence of 3 mM Hg these hyphae displayed spiral growth. Zn and Cd had no effect on border fractal dimension. We suggest that all of the metals slow growth and that Cu, Co and Hg may also disrupt the relationship between tip growth and branching at the edge of the mycelium.

Hyperpolarisation-activated calcium currents found only in cells from the elongation zone of Arabidopsis thaliana roots: Kiegle E, Gilliham M, Haselhoff J, Tester M: Plant J 2000, 21:225–229

Electrical currents are generated by tip-growing cells and have been proposed to be important in the establishment and maintenance of cell polarity. Unlike the situation in all other fungi thus far examined, the electrical current of Allomyces macrogynus normally flows out of the hyphal tip, not into it. In this organism, as with another chytrid Blastocladiella emersonii, the electrical current enters the rhizoids. Extracellular ion-selective microelectrodes detected local acidification of the medium around hyphal apices, but no local accumulation of K+ or Ca2+ at this site. These results suggest that the outward current is generated by H+ efflux, not K+ or Ca2+ efflux. The hyphae of this fungus were found to be weakly chemotropic, in contrast to the rhizoids which were strongly chemotropic. Rhizoid growth was also directed strongly towards the anode of an applied electrical field; the hyphae were directed to the cathode. Chemotropism and galvanotropism are therefore unrelated. We suggest that, as with other chytridomycetes, the rhizoid of A. macrogynus is an organ specializing in nutrient transport. Since transport is often coupled to proton symport the inward electrical current at rhizoids may be the consequence of localized proton-driven solute transport. In hyphae of the oomycete Achlya bisexualis inward current, localized nutrient transport and chemotropic behaviour again are correlated. We conclude that for water moulds, inward currents indicate zones of localized nutrient transport. The current is therefore incidental to the process of tip growth.

Inwardly directed ionic currents of Allomyces macrogynus and other water moulds indicate sites of proton-driven nutrient transport but are incidental to tip growth

Electrical currents are generated by tip-growing cells and have been proposed to be important in the establishment and maintenance of cell polarity. Unlike the situation in all other fungi thus far examined, the electrical current of Allomyces macrogynus normally flows out of the hyphal tip, not into it. In this organism, as with another chytrid Blastocladiella emersonii , the electrical current enters the rhizoids. Extracellular ion-selective microelectrodes detected local acidification of the medium around hyphal apices, but no local accumulation of K + or Ca 2+ at this site. These results suggest that the outward current is generated by H + efflux, not K + or Ca 2+ efflux. The hyphae of this fungus were found to be weakly chemotropic, in contrast to the rhizoids which were strongly chemotropic. Rhizoid growth was also directed strongly towards the anode of an applied electrical field; the hyphae were directed to the cathode. Chemotropism and galvanotropism are therefore unrelated. We suggest that, as with other chytridomycetes, the rhizoid of A. macrogynus is an organ specializing in nutrient transport. Since transport is often coupled to proton symport the inward electrical current at rhizoids may be the consequence of localized proton-driven solute transport. In hyphae of the oomycete Achlya bisexualis inward current, localized nutrient transport and chemotropic behaviour again are correlated. We conclude that for water moulds, inward currents indicate zones of localized nutrient transport. The current is therefore incidental to the process of tip growth.

Evidence for Diploidy inAchlya BisexualisBased on Inheritance of Cycloheximide Resistance

In a study of a cycloheximide-resistant mutant of the oomycete Achlya bisexualis Coker & Couch two important observations have been made. The first concerns the inheritance of cycloheximide resistance and the second is the position of meiosis in the life cycle of this organism. Induced cycloheximide-resistant mutants are known in several species of fungi. The inheritance of cycloheximide resistance in Saccharomyces cerevisiae Hansen was shown to be controlled by six genes, four of which were dominant and two recessive (Wilkie and Lee, 1965). Additional modifier genes were also hypothesized. In both Physarum polycephalum Schw. (Haugli et al., 1972) and Neurospora crassa Shear & Dodge (Hsu, 1963) resistance was shown to be controlled by two loci. In