Mycology: unravelling the riddle of the filamentous fungi

The fungi are perhaps the least understood of the multicellular organisms, despite being almost ubiquitous in nature. An international team, coordinated by Professor Dr Reinhard Fischer of the Karlsruhe Institute of Technology, Germany, and Professor Dr Meritxell Riquelme from the Centro de Investigación Cientifíca y de Educación Superior de Ensenada, Mexico, is leading attempts to understand the growth and development of these remarkable organisms, shedding light on their medical and ecological applications.

Close-up images of a colony of N. crassa. The aerial mycelium shows the interwoven fine hyphae. Carotene stains the mycelium orange. Pictures taken by Rosa Aurelia Fajardo Somera from KIT.

There may be as many as five million species of fungi worldwide — many more than there are plants. The vast majority of these little-understood organisms are the ‘filamentous fungi,’ named because they are composed of a web of filaments called ‘hyphae’. The work of Professor Fischer, Professor Riquleme and their co-workers focuses on how these filaments grow, indefinitely, by extension at each microscopic tip, to form huge networks called ‘mycelia’.

Underrated organisms
Despite their lowly appearance, the filamentous fungi are crucial to the functioning of natural ecosystems. Alongside bacteria, they are the main agents responsible for decomposing dead organic matter, making its chemical components available to the next generation of organisms. What is perhaps less well-known is that they play a vital role, not only in generating nutrients, but also in plant nutrient uptake: every metre of plant root in the soil is associated with roughly a kilometre of symbiotic fungal hyphae, known as ‘mycorrhiza’, which take up nutrients and pass them to the plant.

The diverse and enlightening findings of this high-profile programme have implications far beyond the fungal kingdom.

Filamentous fungi are important pathogens of crop plants, and in a few cases cause serious human disease, particularly in the immunocompromised. They have also been harnessed for biotechnological uses, including crucially in the production of antibiotics such as penicillin, other medicines, citric acid, and foods such as soy sauce and cheese. To scientists, fungi are also important due to the similarity of their cells to human cells, making them ideal models to study various aspects of cell function.

With the help of the jellyfish green fluorescent protein (GFP) researchers visualised the microtubule cytoskeleton in Aspergillus nidulans. Round spores produced long hyphae, and microtubules are visible as long filaments in the cells. They serve as tracks for intracellular traffic. Picture taken by Minoas Evangelinos, KIT

Professor Fischer, Professor Riquelme and their colleagues, with funding from the Deutsche Forschungsgemeinschaft and CONACYT, are studying a host of questions surrounding the growth and development of filamentous fungi. Using multiple species and state-of-the-art microscopy and molecular biological methods, they are enhancing our understanding of the mechanisms by which these intriguing and important organisms grow and differentiate.

Keeping pace with growth
At the tip of each fungal hypha lies a region of active growth. Here, membrane-bound particles (vesicles) containing the raw materials for building new cell walls and membranes — proteins, lipids and other organic molecules, as well as catalytic enzymes — fuse with the cell’s boundary membrane, releasing their precious cargo. However, the highly polarised positioning of this region poses challenges for the fungus. Firstly, how can they transport adequate quantities of these materials to the tip in order to keep up with the rate of growth? Secondly, with such rapid growth occurring at the tip, how does the hypha maintain a stable marker of exactly where, and in what direction, growth is to occur?

The logistics of transporting materials to the actively-growing hyphal tip are being elucidated by Michael Feldbrügge’s lab at Heinrich Heine University, Düsseldorf, Germany. The length of the hyphae is traversed by a skeleton of fine tubes called microtubules, along which vesicles and their contents travel, facilitated by proteins acting as motors. Feldbrügge has also found that these provide transport routes for molecules of messenger RNA, which translate the genetic information in DNA into functional proteins. Crucially, this means that protein production can be precisely targeted to specific regions within a cell without having to transport large quantities of the proteins themselves.

In this experiment two enzymes required for cell wall synthesis were visualised in hyphae of Neurospora crassa using two different coloured fluorescent proteins. Whereas an endoglucanase enzyme (BGT-2) localises to the plasma membrane, a chitin synthase (CHS-1) accumulates first in a structure called “Spitzenkörper” before it is secreted. Confocal Laser scanning microscopy images obtained by Dr Leonora Martínez-Núñez, CICESE.

In answer to the second question, Prof Fischer himself, working with Prof Norio Takeshita, has discovered that in the filamentous fungus, Aspergillus nidulans, molecules of a special protein — TeaR — located at the tip of the hyphae, mark the zone in which active growth is taking place. Using advanced, super-resolution microscopy techniques to visualise the activities of living cells in real time, they showed that the cluster of TeaR molecules at the hyphal tip is repeatedly dispersed and reassembled with newly-arriving TeaRs, maintaining an indicator that tells the machinery of the cell where to build new tissue.

State-of-the-art microscopy and molecular biological methods are enhancing our understanding of the mechanisms by which these intriguing and important organisms grow and differentiate.

A further project, led by Prof Meritxell Riquelme is shedding light on the precise nature of the transport of vesicles found at the growing hyphal tip. Remarkably, it turns out that there are separate populations of differently-sized vesicles carrying different enzymes for building various components of the fungal cell wall.

From repair to reprogramming
In addition to spontaneous growth, organisms also need to repair themselves from damage, and research into filamentous fungi is shedding light onto how this may be achieved. Prof Alfredo Herrera-Estrella at the Laboratorio Nacional de Genómica para la Biodiversidad, Guanajuato, Mexico, and colleagues, use the fungus Trichoderma atroviride, a biocontrol agent, in their work. They have shown, using modern genetic approaches, that injury results in the production of highly reactive and damaging molecules known as ‘reactive oxygen species (ROS)’, stimulating the formation of reproductive structures. This molecular pathway promotes cell differentiation and regeneration in the face of damage and — using ROS as signals — could be shared with both plants and animals. Therefore, understanding it could have important applications in medicine.

Also working at the Karlsruhe Institute, Prof Natalia Requena is studying how filamentous fungi interact with plants when forming arbuscular mycorrhizal symbioses, which improve the plants’ supply of phosphate, sulphur and nitrogen in return for carbohydrates. Within roots, the fungi form specialised structures known as ‘arbuscules’ to allow the nutrient exchange between symbiotic partners. Arbuscule formation, however, requires the fungus to both circumvent plants’ natural defences and to emit signals that actively reprogramme the plant cells to accommodate arbuscules. As molecular biology advances, and the genomes of filamentous fungi are sequenced, our understanding of the precise molecular nature of these interactions will unfold.

From fungi to further afield
The diverse and enlightening findings of this high-profile programme have implications far beyond the fungal kingdom. The methods used — particularly novel ways of imaging microscopic and rapidly-changing structures — have the potential to revolutionise studies of subcellular processes across the living world. Professor Fischer and his collaborators are finally bringing the filamentous fungi from the soil firmly into the limelight.