Inncelly Experimentation Chambers: A novel design for sample handling and live-cell imaging of biological interactions
Biology
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Dr Alexander Lichius and his fellow scientists from the University of Innsbruck, Austria, develop a novel technology – the inncelly experimentation chambers – for live-cell imaging of biological cultures. They use the chambers to explore the fungal cell biology of mycoparasites, including the use of fluorescent molecules, such as CRIB reporters. The research team tracks fundamental cell processes, such as cell polarity of the organisms, which regulate fungal-fungal and fungal-plant interactions. The technology is available for a variety of microscopy types, can be integrated with other physiological measurements and is thus applicable to a range of study fields.
Dr Alexander Lichius and his colleagues from the University of Innsbruck, Austria, have introduced a completely new design of experimental equipment: the inncelly experimentation chambers. They use the chambers in their investigation of fungus-fungus-plant interactions to shed light on how mycoparasitic fungi grow and protect plants from fungal pathogens.
Fungi diversify immensely in their lifestyles: from cooking mushrooms, through the green and black mould we see on products, to single-celled organisms, such as yeast, used for fermentation and bakery. Many species of fungi live in the soil and most have symbiotic or pathogenic relationships with plants.
Symbiotic fungi interact with the plant indirectly through the rhizosphere around the roots, or as so-called endophytes in direct cellular contact. Some species can even parasitise other fungi. This process is called mycoparasitism. Certain mycoparasites are widely used as biofungicide (also termed biocontrol fungi) for crop plant protection against pathogenic fungi, and thus represent an important substitute for chemical fungicides that are harmful to the environment and human health.
“The sampling usually caused some destruction to the organisation of the fungal mycelium, which was eliminated in this new chamber design.”
In order to more closely examine the mycoparasitic relationships, scientists have been using Trichoderma fungi as model organisms for a long time. In their studies, Dr Lichius and colleagues are investigating the mechanisms by which distinct Trichoderma species locate their prey through different chemical molecules and enzymes that are exchanged through complex sequences of signalling pathways. The researchers also use specific fluorescent molecules, such as CRIB reporters, to track the interaction stages.
Trichoderma biology Trichoderma species are useful organisms as they stimulate plant root and shoot growth, provide systemic resistance against pathogenic attacks, and even function as a rapid composter. A lot of Trichoderma species antagonise and – in some cases – directly parasitise other fungi. These mycoparasites detect other fungi via chemical sensing and then establish a direct physical interaction with the prey, eventually leading to its killing.
Fig 1. CRIB reporters allow tracking of fungal interactions on the cellular level. (A) Petri dish confrontation culture between the plant pathogen Fusarium oxysporum (purple) and the mycoparasite Trichoderma asperellum (green). (B) Close up of the interaction zone. (C) Microcolony confrontation culture in an inncelly ib01 chamber provides superior live-cell imaging conditions. Scale bars, 1 cm. (D) CRIB reporters (arrowheads) visualise mycoparasitic hyphae (green) attacking the prey fungus (yellow). (E) Time projection of the dynamic displacement of GTPase activity in the growing tip apex (arrowhead) and the Spitzenkörper (Spk, arrow). Scale bars, 10 μm.
First, Trichoderma detects specific chemical compounds released by the prey fungus into the soil with its hyphae (which are the tubular, branching cells that drive extension of the colony). The mycoparasite releases an increased amount of cell wall degrading enzymes, which are aimed at the prey fungus. The prey’s cell wall then thins and releases even more chemoattractants, which guide the mycoparasite towards it.
After the mycoparasite has located its prey, it establishes direct physical contact by branching, coiling, or even penetration into the prey cells. The branched morphology as well as cell fusion of the mycoparasitic hyphae is key to exchanging signals within the hyphal network (also termed the mycelium). These signals are crucial for regulating the mycoparasitic reaction of the whole colony towards its prey.
Fig 2. The inncelly basic chamber. (A) The ib01 chamber comprises a stamp and base plate and a lid. A glass cover slip carries the agar growth medium. A lid with spacers guarantees sterile yet aerated incubation. (B) The stamp plate allows easy withdrawal of the sample. (C) A microcolony confrontation culture is directly transferred from the ib01 onto the microscope stage (D).
Hyphal growth in response to signal exchange is regulated by the polarity apparatus within the hyphal tip of filamentous fungi. Two types of enzymes – the so-called GTPases Cdc42 and Rac1 – are master regulators of polarised tip growth. They also control other crucial cell processes, such as spore germination and hyphal morphogenesis, including the fusion between fungal cells, changes in the cytoskeleton, and the transport of molecules in and out of the cells. Playing such an essential role, Cdc42 and Rac1 are great for tracking the development of the mycoparasitic lifecycle and fungus-fungus-plant interactions.
Dr Lichius’ research team recently established that these enzymes are involved in regulating the attack or avoidance reaction of Trichoderma in the presence of other fungi. The team used specific fluorescent CRIB (Cdc42-Rac-Interactive-Binding) reporters, to track how the activity of their target GTPases changes as tip growth of the mycoparasite develops in response to different plant-pathogenic prey fungi (Fig. 1).
Upgrading the current technology
The Innsbruck scientists developed their new experimental design to maximise the results of their latest studies. They needed to develop chambers in which to culture the focal fungal species without inducing any stress on the cells through injury or handling. Until now, the sampling usually caused some level of destruction to the natural organisation of the fungal mycelium, which was eliminated in this new chamber design.
“Simple to design, make and optimise, these chambers are a great method for increasing the efficiency of the experimental trials through a standardised sample preparation.”
The lack of such a basic commercially available product inspired the creation of inncelly experimentation chambers. Simple to design, make and optimise, using CAD software and a 3D printer, these chambers are a great method for increasing the efficiency of the experimental trials through a standardised cultivation and sample preparation procedure.
The chambers provide both aerobic and sterile incubation of the cultures. The handling of the samples does not affect or stress the organisms in any way, and the reduced result variation improves the reproducibility of the experiments. Moreover, the chambers are available for a range of the highest quality optics (these include bright-field, DIC, wide-field deconvolution, and confocal fluorescence microscopy), and due to the high flexibility of 3D printing, they can be readily optimised and modified to incorporate additional live-cell physiological measurements, such as light.
The chambers in practice
Dr Alexander Lichius and his team so far developed two types of experimental chambers: the inncelly ib01 and the inncelly il30. Both chamber types are specifically designed for live-cell cultures. The inncelly basic 01 is the chamber for long-term observation and examination of the physical and chemical interactions between organisms, usually growing on solid nutrient media (Fig. 2). It is suitable for filamentous fungi, seedlings, or arbuscular mycorrhizal fungi, and allows for a continuous focus-stable imaging for more than 10 hours and repeated investigations of the samples up to 72 hours after preparation. These chambers are also applicable for studying the molecular and cellular level of the interactions between crop plants, fungal pathogens, and mycoparasitic fungi in their role as biocontrol organisms.
For instance, Dr Lichius and colleagues used this chamber to investigate the interactions between the mycoparasite Trichoderma atroviride (T. atroviride) and one of its preys, the plant-pathogenic grey mould fungus Botrytis cinerea. Continuous imaging sessions in high resolution showed that the interactions between the two species take between 14 and 18 hours from the initial stage of chemical sensing to the physical contact and eventual killing of the prey.
Fig 3. The inncelly light chamber. (A) The geometry and specialised lid of the il30 chamber are adapted for the application of narrow-bandwidth light filters. (B) Removal of the filter cap uncovers the filter for the desired light exposure of the sample. (C) A microcolony culture is transferred from the il30 chamber onto the microscope stage within seconds (D).
The inncelly light chambers provide a tightly regulated light environment for the cultured cells (Fig. 3). They contain a specific filter layer, letting in only the desired wavelengths of light for the desired exposure time. The layer can filter wavelengths with 10 nm accuracy, in the range between 310 and 900 nm, covering the near-UV, visible and infrared light spectrum. These chambers are useful for working with organisms that interact in light-sensitive conditions and for understanding light-dependent cellular processes.
For example, the Innsbruck University scientists are using the il30 chamber to track mycoparasite-pathogen interactions, which usually occur around the root system of the plants, in the soil, where light is scarce or absent. It is crucial in this instance that the light quality is strictly regulated, as the defence mechanism of some species of fungal plant pathogens are weakened in the absence of particular wavelengths.
The interactions between T. atroviride and the plant pathogens Fusarium oxysporum and Fusarium graminearum are examples of such light-regulated effects on prey defence. Under yellow or red light, or in complete darkness, defensive toxin production is much reduced in both Fusarium species making it easy for the mycoparasite to defeat its prey.
Fig 4. Application examples of wavelength-dependent cellular responses. Only blue light between 455-465 nm properly induces (A) the formation of green spores in T. atroviride and (B) nuclear import of the GFP-labelled blue light receptor VVD in N. crassa (Chen et al., 2010. PNAS 107(38)). (C) Image quantification reveals that GFP-VVD shuttling occurs almost twice as efficiently in response to 455-465 nm compared to 425-435 nm, and that the inducing effect is additive under white light.
Moreover, having such precise light filtering showed that the formation of asexual spores in T. atroviride is triggered by a specific fraction of blue light (Fig. 4A). It had previously been shown that the spores form above ground, in light, and where wind can distribute them later. However, now Dr Lichius and colleagues showed that only light with a wavelength of 455-465 nm properly induces spore formation in Trichoderma, whereas other wavelengths trigger weak or no reaction at all.
Another experiment using the same light chambers illustrates that a specific photoreceptor in a different species of filamentous fungus, the orange bread mould Neurspora crassa, is also much more responsive to light between 455-465 nm, compared to 425-435 nm (Fig. 4B). Overall, inncelly experimentation chambers provide precise and versatile approaches to examine interactions in a wide range of biological processes.
Next steps
Such a simple and elegant solution might greatly benefit the environment by saving on materials and reducing plastic waste, given their fabrication from biopolymers and the multiple uses of each chamber. The research team and its associated spin-off company is dedicated to developing further chambers that can suit various types of study in the agricultural, pharmaceutical, or biotechnological sectors. They will be adjusted to measure different physiological factors, such as light, secretion, diffusion, or even production of volatile compounds. In addition, a development pipeline for customised products, according to specific client requirements, is planned to be established in the near future.
What materials can the chambers be made of? Might the different materials have different effects on the culture development?
Generally, the full range of 3D printing polymers can be used to manufacture inncelly chambers and to meet budget and experimentation requirements. Biocompatibility and sterilisation are important features for biological applications. Therefore, materials that comply with food-safety certifications and do not leak deleterious substances into the culture medium are the first choice. Secondly, highly chemical, UV or heat resistant polymers that can be sterilised by various methods are preferred. Finally, fabrication with biopolymers produced from renewable, plant-based resources reduce the dissipation of non-biodegradable plastic and contribute to sustainability.
References
Moreno-Ruiz, D., Salzmann, L., Fricker, M.D., et al. (2021). Stress-activated protein kinase signalling regulates mycoparasitic hyphal-hyphal interactions in Trichoderma atroviride. Journal of Fungi, 7(5), 365. Available at: https://doi.org/10.3390/jof7050365
Karlsson, M., Atanasova, L., Jensen, D.F., and Zeilinger, S. (2017). Necrotrophic Mycoparasites and Their Genomes, 1005-1026. In: Heitman, J., Howlett, B., Crous, P., et al. (Eds). The Fungal Kingdom. ASM Press, Washington, DC. Available at: https://doi.org/10.1128/microbiolspec.FUNK-0016-2016.
Zeilinger, S., Gupta, V.J., Dahms, T.E.S., et al. (2016). Friends or foes? Emerging insights from fungal interactions with plants. FEMS Microbiology Reviews, 40(2), 182-207. Available at: https://doi.org/10.1093/femsre/fuv045
Lichius, A., Goryachev, A.B., Fricker, M.D., et al. (2014). CDC-42 and RAC-1 regulate opposite chemotropisms in Neurospora crassa. Journal of Cell Sciences, 127, 1953-1956. Available at: https://doi.org/10.1242/jcs.141630
Dr Alexander Lichius and colleagues have developed the inncelly experimentation chambers to closely study the fungal cell biology of mycoparasites in fungus-fungus-plant interactions and aim on expanding this technology to other biological systems with their spin-off company inncellys GmbH.
Funding
Tiroler Wissenschaftsförderung (TWF)
Förderkreis 1669, University of Innsbruck
Collaborators
Prof Susanne Zeilinger, University of Innsbruck, AT
Prof Mark D. Fricker, University of Oxford, UK
Prof Lionel Dupuy, Neiker, ES
Prof Antonio Di Pietro, University of Cordoba, ES
Prof Andrew Goryachev, University of Edinburgh, UK
Acknowledgement
Dr Lichius thanks his co-inventor MSc Laura Hackl for contributing fungal colony images, and Prof Jay C. Dunlap from Dartmouth Medical School for providing N. crassa GFP-VVD strains.
Bio
Dr Alexander Lichius studied Biology and Biophysics at the Universities of Aachen and Dresden, Germany, before completing his PhD in Fungal Cell Biology at the University of Edinburgh, UK. He was postdoc at CICESE in Baja California, Mexico and at the University of Technology Vienna, Austria. He has been working at the University of Innsbruck since 2016.
Dr Alexander Lichius
Contact
Dr Alexander Lichius
Department of Microbiology
University of Innsbruck
Technikerstrasse 25
Victor-Franz-Hess Haus, 6th Floor,
Room 6/04, 6020 Innsbruck, Austria
