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Mediators in plant pathogenic fungal-bacterial symbiosis

Periodic Reporting for period 1 - FUNBIOSIS (Mediators in plant pathogenic fungal-bacterial symbiosis)

Reporting period: 2018-06-01 to 2020-05-31

Interspecies interactions between fungi and bacteria are highly prevalent in nature and are critically important in a variety of fields including agriculture, food processing, biotechnology, medicine, and dentistry. The study of such bacterial-fungal interactions (BFIs) can provide invaluable information regarding host-pathogen interactions that can be leveraged in understanding and treating important animal and plant diseases.
Research on BFIs has three major benefits for society: the potential discovery of new drugs and biopesticides, the improvement of prevention and treatment against bacterial and fungal infections, and the enhancement of analytical and molecular approaches to study microbial interactions that may pose a risk or may be interesting in other fields, such as the study of microbial communities in oil spills or in extreme ecological environments.
Endosymbiotic BFIs, where the bacterial partner resides within the fungus, are ubiquitous and have been globally documented for more than three decades. While endosymbiosis in animals and plants is exceptionally well studied, only little is known about the occurrence of endosymbiotic bacteria within the fungal kingdom. Historically, such endosymbiotic interactions have been difficult to detect, because the bacteria are “hidden” inside the fungal mycelium. Complicating matters further, many endosymbiotic bacteria are unable to grow in pure culture outside of their host, making their study and manipulation a daunting task.
The biggest challenge in studying the dynamic interactions between filamentous fungi and bacteria is the monitoring of specific interactions between hyphae and bacteria and their spatial organisation. In traditional methods (e.g. macroscale confrontation assays, microscopic imaging) it is not possible to control environmental conditions in a precise and dynamic manner while monitoring the interactions at a microscopic level. One emerging technology to overcome these limitations is microfluidics. These engineered, micron-scale systems enable fungal hyphae to be cultured in micro channels, thus allowing easy imaging at single-cell level. Such platforms are compatible with high-resolution microscopy techniques (e.g. spinning disk confocal) and therefore allow live-cell imaging and long-term, time-lapse microscopy to be conducted with ease. Coupling of microfluidics with high performance liquid chromatography (HPLC), mass spectrometry (MS), and automated image processing will provide opportunities to quantify effector molecules involved in interactions between fungi and bacteria.
An intriguing example of BFIs is the endosymbiosis between the plant-pathogenic zygomycete Rhizopus microsporus and its toxin-producing bacterial endosymbiont Burkholderia rhizoxinica. Some of the unique features that make this symbiosis an attractive model for studying secreted effector molecules central to BFIs are: 1) The presence of the bacterial symbiont can be readily determined by its production of the phytotoxin rhizoxin; 2) R. microsporus can be cured of its bacterial symbiont with antibiotic treatment and the bacteria can be cultured outside of the fungal host, allowing for genetic manipulations of the endosymbiont; and 3) the fungus is unable to sporulate asexually in the absence of the endosymbiont. Re-infecting the cured fungus with cultured bacteria can restore rhizoxin production and the ability to sporulate. FUNBIOSIS aims to speed up the discovery of strategies and effectors used by endosymbiotic bacteria to control their fungal hosts by integrating the emerging technology of microfluidics.
FUNBIOSIS has been involved in several multidisciplinary projects to study microbial interactions, especially bacterial-fungal interactions.
1. Transcription activator-like effectors (TALEs) are a class of protein effectors from plant pathogenic bacteria that directly modify the expression of host plant genes to help bacterial colonisation. We show an unprecedented case where TALEs protect endosymbiotic bacteria from entrapment within fungal hyphae and thus identified a fundamentally new function of this widespread family of effectors. A combination of novel microfluidic devices, fluorescence microscopy, and high-resolution live imaging provided the first real-time snapshot of septa biogenesis in a zygomycete fungus leading to hyphal trapping of endosymbionts incapable of secreting TALEs.
Richter, I., Uzum, Z., Stanley, C.E. Moebius, N., Stinear, T.P. Pidot, S.J. Ferling, I., Hillmann, F., & Hertweck, C. (2020). Secreted TAL effectors protect symbiotic bacteria from entrapment within fungal hyphae. bioRxiv 2020.03.28.013177; doi:
2. The coenzyme F420 is a specialised redox cofactor with a highly negative redox potential. Genome sequencing revealed the presence of F420 biosynthetic genes in B. rhizoxinica. Fluorescence microscopy, high-resolution LC-MS, and structure elucidation by NMR revealed that B. rhizoxinica produces unexpected F420-derivatives (3PG-F420) in symbiosis with its host. Genetic and biochemical studies by the group of Dr. Gerald Lackner at HKI demonstrate that a switch in substrate specificity of the guanylyltransferase CofC is responsible for the biosynthesis of 3PG-F420, suggesting a rerouting event during the evolution of F420 biosynthesis.
Braga, D., Last, D., Hasan, M., Guo, H., Leichnitz, D., Uzum, Z., Richter, I., Schalk, F., Beemelmanns, C., Hertweck, C., & Lackner, G. (2019). Metabolic Pathway Rerouting in Paraburkholderia rhizoxinica Evolved Long-Overlooked Derivatives of Coenzyme F420. ACS Chemical Biology, 14(9), 2088-2094.
3. The group of Prof. Christian Hertweck investigates pathogenic Burkholderia species to discover virulence factors used by these bacteria to infect and eventually kill humans and animals. Unusual cyclopropanol-substituted polyketides were identified in the model organism B. thailandensis. Cell-based assays and a nematode infection model showed that this rare natural product confers cytotoxicity and virulence.
Trottmann, F., Franke, J., Richter, I., Ishida, K., Cyrulies, M., Dahse, H.-M. Regestein, L., & Hertweck, C. (2019). Cyclopropanol Warhead in Malleicyprol Confers Virulence of Human- and Animal-Pathogenic Burkholderia Species. Angewandte Chemie International Edition, 58, 14129.
FUNBIOSIS discovered a fundamentally new function of a widespread effector family, thereby providing new insights into the complex interactions of bacteria and fungi that form a close symbiotic alliance. This mechanism of host control illustrates an unusual survival strategy of endosymbionts in the host that has broader implications, as it may serve as a model to understanding how prokaryotic pathogens and symbionts interact with eukaryotic hosts. FUNBIOSIS has collaborated in a project that led to the discovery of novel derivatives of the rare cofactor F420 involved in bacterial-fungal interactions. The pathway discovered offers an alternative route to the production of F420-dependent enzymes for biocatalysis. The scope of the project has been extended beyond fungal-bacterial interactions due to more collaboration requests. Executed projects include other interactions such as bacteria-nematode, yeast-nematode, and fungal-nematode interactions. One such study led to the discovery of a new family of natural products (cyclopropanol-substituted polyketides) associated with an inter-kingdom interaction.