Periodic Reporting for period 1 - FungalSecrets (The role of plant microbiota in the evolution of fungal pathogens and their repertoires of secreted proteins)
Berichtszeitraum: 2023-05-01 bis 2025-10-31
Fungi are important plant pathogens. They invade plant tissues through natural openings, such as stomata on leaves or wounds on root surfaces. In order to avoid or suppress the plant's defences, fungal pathogens secrete a wide variety of small molecules known as effectors, which interfere with immune responses and promote further host invasion. Many of these secreted molecules are proteins. Until now, these secreted proteins have been studied in the context of molecular plant-pathogen interactions; however, accumulating evidence suggests that many of these proteins play an important role as antimicrobial effectors, antagonising other microorganisms in the host tissue. The aim of this project is to elucidate the antimicrobial properties of secreted proteins in four fungal plant pathogens. We hypothesise that interactions with the host microbiota may be crucial for the successful invasion of plant tissues. Furthermore, we hypothesise that the repertoire of antimicrobial effectors produced by a given pathogen evolves in response to the particular microbiota of the host. To test these hypotheses, we will integrate different types of 'omics' data, machine learning approaches, and experimental methods. We have established culture collections of bacteria and fungi from three plant species, wild relatives of wheat, barley and sugar beet, four plants which are host the four fungal pathogens under study. This enables us to test if effector proteins, produced by the fungal pathogens, can suppress the growth of certain plant-associated microorganisms. For effectors that can suppress bacterial growth, we aim to understand the underlying mechanisms. Moreover, we aim to understand why some plant-associated microorganisms are suppressed by the fungal pathogen. To this end, we use plant experiments in which we propagate plants with defined microbial communities and pathogen mutants lacking a given effector of interest. The project will deliver novel insights into the relevance and diversity of microbial interactions in plants. Furthermore, we will unravel how such microbial interactions shape the evolution and host specificity of fungal plant pathogens.
For the computational predictions, we use already available tools. However, we have incorporated a set of tools in a pipeline which allow us to integrate different types of data to predict protein function and evolution. We have for example conducted a comparative analysis of available tools for protein structure prediction. She has compared predicted structures with experimentally validated protein structures (crystallized protein structures). This has provided us with a robust framework for our analyses for which we have identified limitations of our approach. In brief the main challenge lies in the prediction of small proteins < 100 amino acids. For these the confidence score for the structural prediction is reduced. Furthermore, the computational side of FungalSecrets involve the prediction of putative antimicrobial properties. We are validating antimicrobial functions for a larger set of proteins. For the experimental validation of antimicrobial properties, we have tested different constructs for expression in Pichia. We finally have an expression vector, which allow us to obtain considerable amounts of secreted proteins from the yeast cells using zeosine as a selection marker, not only for yeast transformation, but also for effector expression.
For in planta studies of pathogen-microbiome interactions, we have obtained a large collection of bacteria and fungi from leaves of four plant species (see above). We are currently optimising a synthetic community approach to study the relevance of selected microorganisms on plant-pathogen interactions.
For in planta studies of pathogen-microbiome interactions, we have obtained a large collection of bacteria and fungi from leaves of four plant species (see above). We are currently optimising a synthetic community approach to study the relevance of selected microorganisms on plant-pathogen interactions.
We have predicted the structure of the secretome of the barley-infecting pathogen Zymoseptoria passerinii, and have described the structural relationships of proteins within one fungal genome. We have further used different machine learning approaches to predict the function of the secreted proteins, whereby we mainly focus on proteins predicted to encode antimicrobial proteins and/or immune-interring proteins. One of the most interesting discoveries has been the large number of secreted proteins which are predicted to encode antimicrobial proteins. This includes a cluster of proteins annotated as killer proteins (KP) which we use to test the robustness of the functional predictions.
Clusters of Killer Proteins (KP), produced by Z. passerinii are predicted to include both immune suppressing and antimicrobial proteins. This could suggest that conserved proteins folds have served as a scaffold for functional diversification and innovation. Our preliminary results suggest that some of KP4 and KP6 proteins, as predicted, have anti-fungal or anti-bacterial functions while others have immune suppressing properties.
While we have been focusing on antagonistic interactions between the fungal pathogens and plant-associated microorganisms, we have frequently observed another phenomenon: Bacteria which grow stronger in the vicinity of Zymoseptoria pathogens. This observation prompt us to ask the question if the fungus secretes i) compounds that can serve as nutrient source of bacteria or ii) enzymes which modulates the growth substrates in favour of bacterial proliferation. We discovered that the bacterial proliferation only occurs in media containing sucrose. This is consistent with the secretion of the enzyme invertase by Z. tritici. Invertase convert sucrose into glucose and fructose which can be more readably taken up and metabolised by bacteria that do not themselves encode the enzyme invertase. We conclude that sugar metabolism by Z. tritici creates a favourable environment for some bacteria also inhabiting the plant. The discovery points to a process which accelerates microbial competition in plant tissues and demonstrate a mechanism whereby pathogens can promote the proliferation of other microorganisms.
Clusters of Killer Proteins (KP), produced by Z. passerinii are predicted to include both immune suppressing and antimicrobial proteins. This could suggest that conserved proteins folds have served as a scaffold for functional diversification and innovation. Our preliminary results suggest that some of KP4 and KP6 proteins, as predicted, have anti-fungal or anti-bacterial functions while others have immune suppressing properties.
While we have been focusing on antagonistic interactions between the fungal pathogens and plant-associated microorganisms, we have frequently observed another phenomenon: Bacteria which grow stronger in the vicinity of Zymoseptoria pathogens. This observation prompt us to ask the question if the fungus secretes i) compounds that can serve as nutrient source of bacteria or ii) enzymes which modulates the growth substrates in favour of bacterial proliferation. We discovered that the bacterial proliferation only occurs in media containing sucrose. This is consistent with the secretion of the enzyme invertase by Z. tritici. Invertase convert sucrose into glucose and fructose which can be more readably taken up and metabolised by bacteria that do not themselves encode the enzyme invertase. We conclude that sugar metabolism by Z. tritici creates a favourable environment for some bacteria also inhabiting the plant. The discovery points to a process which accelerates microbial competition in plant tissues and demonstrate a mechanism whereby pathogens can promote the proliferation of other microorganisms.