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In vitro xylem induction system is a basic tool in physiological, biochemical, and molecular studies of secondary cell wall formation, lignin biosynthesis and deposition associated with tracheary element formation. Eucalyptus bosistoana is a Class 1 durable hardwood tree species, selected by the New Zealand Dryland Forest Initiative for good quality wood and high adaptability to the NZ growing conditions. Xylogenic E. bosistoana callus culture was established and up to 40% of the callus cells were xylem-like cells (XLCs) which may have differentiated from small, cytoplasmically dense or compact dividing, and exhibited increased lignin contents during culture. The eucalyptus XLCs showed diverse sizes, patterns of secondary cell wall thickenings similar to the xylem cells in the young shoots and organized development including cell–cell connections of the XLCs to form xylem strands. This is the first report of the organized development of XLCs in E. bosistoana callus culture.

β-Cyclodextrin (β-CD) and derivatives are approved therapeutics in30 clinical settings. β-CDs have also shown promise as therapeutics for treatment of some lysosomal storage disorders, such as Niemann-Pick disease type C, and other disease states which involve metabolite accumulation in the lysosome. In these cases, β-CD activity relies on transport to the lysosome, wherein it can bind hydrophobic substrate and effect extraction. The post-translational attachment of N-glycans terminated in mannose-6-phosphate (M6P) residues is the predominant method by which lysosomal enzymes are targeted to the lysosome. In this work we covalently attach a synthetic biantennary bis-M6P-terminated N-glycan to β-CD and study the effect of the added glycans in a mammalian cell line. The formation of a host guest complex with a Cy5 fluorophore allows study of both cellular internalisation and transport to the lysosome by fluorescence microscopy. Results indicate that the rates of both internalisation and lysosomal transport are increased by the attachment of M6P-glycans to β-CD, indicating that M6P-glycan conjugation may improve the therapeutic effectiveness of β-CD for the treatment of disorders involving hydrophobic metabolite accumulation in the lysosome.

Pathogenic fungi and oomycetes give rise to a significant number of animal and plant diseases. While the spread of these pathogenic microorganisms is increasing globally, emerging resistance to antifungal drugs is making associated diseases more difficult to treat. High-throughput screening (HTS) and new developments in lab-on-a-chip (LOC) platforms promise to aid the discovery of urgently required new control strategies and anti-fungal/oomycete drugs. In this review, we summarize existing HTS and emergent LOC approaches in the context of infection strategies and invasive growth exhibited by these microorganisms. To aid this, we introduce key biological aspects and review existing HTS platforms based on both conventional and LOC techniques. We then provide an in-depth discussion of more specialized LOC platforms for force measurements on hyphae and to study electro- and chemotaxis in spores, approaches which have the potential to aid the discovery of alternative drug targets on future HTS platforms. Finally, we conclude with a brief discussion of the technical developments required to improve the uptake of these platforms into the general laboratory environment.

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.

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]

Oomycetes and fungi are microorganisms whose pathogenic (invasive) growth can cause diseases that are responsible for significant ecological and economic losses. Such growth requires the generation of a protrusive force, the magnitude and direction of which involves a balance between turgor pressure and localised yielding of the cell wall and the cytoskeleton. To study invasive growth in individual hyphae we have developed a lab-on-a-chip platform with integrated force-sensors based on elastomeric polydimethylsiloxane (PDMS) micro-pillars. With this platform we are able to measure protrusive force (both magnitude and direction) and hyphal morphology. To show the usefulness of the platform, the oomycete Achlya bisexualis was inoculated and grown on a chip. Growth of individual hyphae into a micro-pillar revealed a maximum total force of 10 μN at the hyphal tip. The chips had no discernible effect on hyphal growth rates, but hyphae were slightly thinner in the channels on the chips compared to those on agar plates. When the hyphae contacted the pillars tip extension decreased while tip width increased. A. bisexualis hyphae were observed to reorient their growth direction if they were not able to bend and effectively grow over the pillars. Estimates of the pressure exerted on a pillar were 0.09 MPa, which given earlier measures of turgor of 0.65 MPa would indicate low compliance of the cell wall. The platform is adaptable to numerous cells and organisms that exhibit tip-growth. It provides a useful tool to begin to unravel the molecular mechanisms that underlie the generation of a protrusive force.

This paper reports a triple-layer, polydimethylsiloxane (PDMS)-based lab-on-a-chip platform combining the capture and culture of individual oomycete zoospores with integrated force sensing on germinated hyphae. The platform enables the concurrent study of cell-to-cell variability in hyphal growth and protrusive force generation. To demonstrate the applicability of the platform, individual zoospores of the oomycete Achlya bisexualis were trapped by a constriction structure, cultured on the device and the micro-Newton forces exerted by hyphae measured by tracking the deflection of elastomeric micropillars. The platform provides a new tool to help understand protrusive growth on a single cell level.

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.

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 platform design, the fabrication process and photoresist optimization required to adapt the microfluidic platform to relatively thin hyphae. To demonstrate the applicability of the platform, the fungus Neurospora crassa was cultured on PDMS chip and the force exerted by individual hyphae measured via the deflection of the micropillars. 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.