Elastomeric micropillar arrays for the study of protrusive forces in hyphal invasion

Fungi and Oomycetes are microorganisms that can be pathogenic and grow invasively causing significant economic losses and diseases1. • These organisms grow by extending the cell at the tip. This involves turgor pressure, cell wall yielding and a dynamic cytoskeleton, giving rise to a protrusive force2,3. •A Lab-on-a-Chip platform, with integrated force sensor based on elastomeric micro-pillars, is allowing us to study the molecular mechanisms which enable the generation of protrusive force at the tip of invasively-growing hyphae. •A maximum force of 16 μN was measured for the oomycete Achlya bisexualis cultured on the chip

Novel Bi-Directional Dual-Flow-Rootchip to Study Effects of Osmotic Stress On Calcium Signalling in Arabidopsis Roots

Being able to detect and respond to abiotic and biotic stresses is fundamental for plant growth and survival. However, understanding of signal transduction within the root remains limited. To help shed light on these processes, we have developed a bidirectional-dual‐flow‐RootChip (bi-dfRC), which adds bidirectional stimulation to the existing asymmetric laminar flow root perfusion platforms. In this paper we show design, fabrication and characterisation of the bi-dfRC, as well as growth of wildtype and Ca2+ indicator (G-CaMP3) Arabidopsis thaliana plants on the platform. Applicability of the bi-dfRC is further demonstrated by probing the dynamic response of Arabidopsis roots to simulated drought stress effects via a fluorescent Ca2+ sensor in a variety of combinations and spatial orientations. The latter enables the tracking of growth, localisation, and quantity in response to bidirectional stimulation in real time at a cellular level

Diverse forms of xylem-like cells and strand formation in Xylogenic Eucalyptus bosistoana callus culture

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

Microfluidic Platform to Study Electric Field Based Root Targeting by Pathogenic Zoospores

This paper reports the fabrication and application of a microfluidic Lab-on-a-Chip platform to study the electrotactic movements of pathogenic microorganisms. The movement of the pathogens in response to electric fields are one way in which they are thought to locate their hosts. Design and fabrication of the platform, and associated micro-electronics are described. The platform contains arrays of micro-electrodes that generate an electric field of defined strength in a micro-chamber into which feed inlet and outlet channels for entry and exit of media and microorganisms. To demonstrate applicability of the platform, motile zoospores of the pathogenic oomycete Phytophthora nicotianae were seeded in the inlet and a voltage was applied to investigate the electrotactic responses of the zoospores. This platform offers a unique opportunity to study electrotactic movements that may be responsible for the ability of the pathogens to locate and invade host tissue

A monolithic polydimethylsiloxane platform for zoospore Capture, germination and single hypha force sensing

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

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

© 1992-2012 IEEE. 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

Parallel screening of single zoospore germination and germ tube protrusive forces

This paper reports the parallel screening of zoospore germination and protrusive forces of germ tubes on a labon-a-chip platform, integrating valve-based single zoospore compartmentalization with micropillar force sensing. Individually-controllable, normally-closed microvalves were optimized to facilitate zoospore compartmentalization in measurement channels containing traps and sensing pillars. Performance was evaluated using microspheres and zoospores of the oomycete Achlya bisexualis. By parallelizing the screening, the platform will support fundamental studies and aid in the discovery of new compounds to control fungal and oomycete pathogens

On-chip measurement of protrusive force exerted by single hyphal tips of pathogenic microorganisms

Invasive growth is a process used by fungi and oomycetes to find sources of nutrients. To study the protrusive forces generated as part of this process, we have developed a Lab-on-a-Chip platform capable of measuring forces exerted by individual hyphal tips during hyphal growth. Force measurement was demonstrated by culturing the oomycete Achlya bisexualis on-chip. Protrusive forces of 7.5 μN and bending forces of 19 μN were recorded for this organism. The platform provides a useful tool to better understand the mechanisms enabling fungi and oomycetes to grow invasively

An elastomeric micropillar platform for the study of protrusive forces in hyphal invasion

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 uN 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