Berberine bridge enzyme-like proteins as key virulence factors in plant pathogens
Lead Research Organisation:
James Hutton Institute
Department Name: Cell & Molecular Sciences
Abstract
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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.
