Berberine bridge enzyme-like proteins as key virulence factors in plant pathogens
Lead Research Organisation:
University of York
Department Name: Biology
Abstract
Plant pathogens cause $300 billion worth of damage to global food production annually. The development of sustainable and targeted disease control approaches underpins global food security and positively impacts public health, social stability and environmental biodiversity. Together, fungi and oomycetes are the most destructive pathogens in modern agriculture and represent a persistent threat to global food security. The oomycete Phytophthora infestans and the fungus Botrytis cinerea are major agricultural pathogens and model species to investigate the molecular mechanisms driving plant-pathogen interactions.
The plant cell wall is the first protective barrier against pathogens and is composed of a complex network of cellulose microfibrils embedded in hemicellulose and lignin, plus a layer of pectin forming the bulk of the middle lamella that joins cells together. The plant cell wall structure and molecular composition have driven the evolution of an impressive range of degradative enzymes in both fungi and oomycetes. During infection, plant pathogens produce cell wall degrading enzymes (CWDEs), like glycoside hydrolases, esterases and lyases, to disrupt the plant cell wall and facilitate tissue penetration. Most of these enzymes are yet to be characterised in any detail. As part of the battle against pathogens, plants produce specific inhibitors of CWDEs and use receptors to sense plant cell wall fragments (oligosaccharides) released by the pathogen`s enzymes, and trigger host immunity. This complex arsenal of offensive and defensive mechanisms revolving around plant cell wall polysaccharides testifies to their important roles in plant-pathogen interactions.
We previously discovered a family of lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and showed that they are key virulence factors involved in the degradation of pectin, the most abundant charged polysaccharide in the plant cell wall. More recently, we have identified a large number of secreted, uncharacterised FAD-dependent oxidases called berberine bridge enzyme-like proteins (BBEs), that have expanded in fungal and oomycete plant pathogens and are strongly induced during infection. We have produced one P. infestans BBE in yeast, carried out in vitro activity assays and detected specific oxidative activity on negatively charged pectin fragments (oligogalacturonides). Through activity assays, we observed that purified P. infestans LPMO and BBE work synergistically to degrade homogalacturonan (the backbone of pectin). Silencing of the most expressed BBE-coding gene in P. infestans caused complete loss of pathogenicity on potato leaves, confirming that this enzyme has a central role in pathogenesis. Our transcriptomic data also indicate that, like oomycetes, phytopathogenic fungi have co-opted BBEs as part of their offensive arsenal and the coding genes are co-expressed with numerous GHs involved in the degradation of abundant plant cell wall polysaccharides, again supporting an active role during tissue penetration.
We hypothesise that oomycetes and fungi secrete BBEs to (i) drive plant cell wall degradation by LPMOs and (ii) oxidatively modify oligosaccharide elicitors released during infection, thus preventing their recognition by plant receptors and dampening the activation of the plant defence responses. Elucidating the molecular roles of BBEs during plant infection and their interplay with other virulence factors will help unlock new strategies to combat plant diseases. In this project, we will use gene silencing to reveal the importance of induced BBE genes during host invasion by fungi and oomycetes and assess their feasibility as targets for crop protection. We will produce recombinant forms of BBE proteins, characterise their biochemical activities and structures, and reveal their synergy with co-secreted enzymes. Finally, we will unveil if and how pathogens use BBEs and their products to manipulate the host immune response.
The plant cell wall is the first protective barrier against pathogens and is composed of a complex network of cellulose microfibrils embedded in hemicellulose and lignin, plus a layer of pectin forming the bulk of the middle lamella that joins cells together. The plant cell wall structure and molecular composition have driven the evolution of an impressive range of degradative enzymes in both fungi and oomycetes. During infection, plant pathogens produce cell wall degrading enzymes (CWDEs), like glycoside hydrolases, esterases and lyases, to disrupt the plant cell wall and facilitate tissue penetration. Most of these enzymes are yet to be characterised in any detail. As part of the battle against pathogens, plants produce specific inhibitors of CWDEs and use receptors to sense plant cell wall fragments (oligosaccharides) released by the pathogen`s enzymes, and trigger host immunity. This complex arsenal of offensive and defensive mechanisms revolving around plant cell wall polysaccharides testifies to their important roles in plant-pathogen interactions.
We previously discovered a family of lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and showed that they are key virulence factors involved in the degradation of pectin, the most abundant charged polysaccharide in the plant cell wall. More recently, we have identified a large number of secreted, uncharacterised FAD-dependent oxidases called berberine bridge enzyme-like proteins (BBEs), that have expanded in fungal and oomycete plant pathogens and are strongly induced during infection. We have produced one P. infestans BBE in yeast, carried out in vitro activity assays and detected specific oxidative activity on negatively charged pectin fragments (oligogalacturonides). Through activity assays, we observed that purified P. infestans LPMO and BBE work synergistically to degrade homogalacturonan (the backbone of pectin). Silencing of the most expressed BBE-coding gene in P. infestans caused complete loss of pathogenicity on potato leaves, confirming that this enzyme has a central role in pathogenesis. Our transcriptomic data also indicate that, like oomycetes, phytopathogenic fungi have co-opted BBEs as part of their offensive arsenal and the coding genes are co-expressed with numerous GHs involved in the degradation of abundant plant cell wall polysaccharides, again supporting an active role during tissue penetration.
We hypothesise that oomycetes and fungi secrete BBEs to (i) drive plant cell wall degradation by LPMOs and (ii) oxidatively modify oligosaccharide elicitors released during infection, thus preventing their recognition by plant receptors and dampening the activation of the plant defence responses. Elucidating the molecular roles of BBEs during plant infection and their interplay with other virulence factors will help unlock new strategies to combat plant diseases. In this project, we will use gene silencing to reveal the importance of induced BBE genes during host invasion by fungi and oomycetes and assess their feasibility as targets for crop protection. We will produce recombinant forms of BBE proteins, characterise their biochemical activities and structures, and reveal their synergy with co-secreted enzymes. Finally, we will unveil if and how pathogens use BBEs and their products to manipulate the host immune response.
Technical Summary
This project will uncover the biological roles, biochemical and structural properties of berberine-bridge enzyme-like proteins (BBEs), a broad new group of oxidative virulence factors we have recently identified in fungal and oomycete plant pathogens.
We will perform gene silencing to determine which BBE genes are important in pathogen virulence during plant infection, and if this role changes according to different pathogen lifestyles (biotrophic, hemibiotrophic and necrotrophic). We will use dsRNAi and transgene-mediated gene silencing to knock-down BBE gene expression in three major plant pathogens and score disease symptoms during infection.
We will produce recombinant versions of the most important BBEs identified in the selected plant pathogens and determine their activities and substrate specificities through enzymatic assays and mass spectrometry techniques. X-ray crystallography will unveil structural details about the proteins` active site, interactions with substrates and cofactor binding properties. We will dissect the mechanisms underlying BBE-LPMO interactions through enzymatic studies and electron paramagnetic resonance (EPR) spectroscopy.
We will determine if and how BBE-catalysed oxidation of oligosaccharides suppresses their efficacy as elicitors. Pure BBEs, as well as native and oxidised oligosaccharides, will be tested for their ability to alter plant resistance to subsequent infection, expression of key plant immunity genes, production of reactive oxygen species (ROS) and callose deposition. We will use thermal shift analysis, enzyme-linked immunosorbent assays (ELISAs) and X-ray crystallography to study the molecular interactions between BBE products and the plant WAK1 receptor, which specifically recognises and binds oligogalacturonides, thereby triggering plant immune responses.
We will perform gene silencing to determine which BBE genes are important in pathogen virulence during plant infection, and if this role changes according to different pathogen lifestyles (biotrophic, hemibiotrophic and necrotrophic). We will use dsRNAi and transgene-mediated gene silencing to knock-down BBE gene expression in three major plant pathogens and score disease symptoms during infection.
We will produce recombinant versions of the most important BBEs identified in the selected plant pathogens and determine their activities and substrate specificities through enzymatic assays and mass spectrometry techniques. X-ray crystallography will unveil structural details about the proteins` active site, interactions with substrates and cofactor binding properties. We will dissect the mechanisms underlying BBE-LPMO interactions through enzymatic studies and electron paramagnetic resonance (EPR) spectroscopy.
We will determine if and how BBE-catalysed oxidation of oligosaccharides suppresses their efficacy as elicitors. Pure BBEs, as well as native and oxidised oligosaccharides, will be tested for their ability to alter plant resistance to subsequent infection, expression of key plant immunity genes, production of reactive oxygen species (ROS) and callose deposition. We will use thermal shift analysis, enzyme-linked immunosorbent assays (ELISAs) and X-ray crystallography to study the molecular interactions between BBE products and the plant WAK1 receptor, which specifically recognises and binds oligogalacturonides, thereby triggering plant immune responses.
