Inncelly Experimentation Chambers: Novel imaging of biological interactions

Hello and welcome to Research Pod. Thank you for listening and joining us today. In this episode we will be looking at the research of Dr Alexander Lichius and his colleagues from the University of Innsbruck in Austria. They developed a new technology for live-cell imaging of biological cultures.

Dr Alexander Lichius and his colleagues 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. They represent an important substitute for chemical fungicides that are harmful to the environment and human health.

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.

How do Trichoderma fungi interact with their prey?

Trichoderma species are useful organisms: 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.

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.

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 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.

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 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.

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. Because of the high flexibility of 3D printing, they can be easily 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 ib01 is the chamber for long-term observation and examination of the physical and chemical interactions between organisms, usually growing on solid nutrient media. 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 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.

The inncelly light chambers provide a tightly regulated light environment for the cultured cells. 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 nanometre accuracy, in the range between 310 and 900 nanometre, 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.

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. Here it is crucial 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 Trichoderma 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.

Moreover, having such precise light filtering showed that the formation of asexual spores in Trichoderma atroviride is triggered by a specific fraction of blue light. 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 nanometre 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 Neurospora crassa, is also much more responsive to light between 455-465 nanometre, compared to 425-435 nanometre.

Overall, inncelly experimentation chambers provide precise and versatile approaches to examine interactions in a wide range of biological processes.

The newest development – the inncelly ib02 chamber – combines the advantages of the ib01 and il30 chambers. Inncelly ib02 chambers offer a larger culture volume and can be equipped with 50x50mm light filters – ideal for long-term investigations of light-dependent cellular processes for which the other two chambers are too small.

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.

That’s all for this episode – thanks for listening, and stay subscribed to Research Pod for more of the latest science. See you again soon.