RESUMEN
Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave recalcitrant polysaccharides such as cellulose. Several studies have reported LPMO action in synergy with other carbohydrate-active enzymes (CAZymes) for the degradation of lignocellulosic biomass but direct LPMO action at the plant tissue level remains challenging to investigate. Here, we have developed a MALDI-MS imaging workflow to detect oxidised oligosaccharides released by a cellulose-active LPMO at cellular level on maize tissues. Using this workflow, we imaged LPMO action and gained insight into the spatial variation and relative abundance of oxidised and non-oxidised oligosaccharides. We reveal a targeted action of the LPMO related to the composition and organisation of plant cell walls.
Asunto(s)
Oxigenasas de Función Mixta , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Zea mays , Zea mays/química , Oxigenasas de Función Mixta/metabolismo , Oxigenasas de Función Mixta/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Celulosa/química , Celulosa/metabolismo , Pared Celular/química , Pared Celular/metabolismo , Oligosacáridos/química , Oligosacáridos/metabolismo , Lignina/química , Lignina/metabolismo , Oxidación-Reducción , Polisacáridos/química , Polisacáridos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismoRESUMEN
Mutualistic symbioses have contributed to major transitions in the evolution of life. Here, we investigate the evolutionary history and the molecular innovations at the origin of lichens, which are a symbiosis established between fungi and green algae or cyanobacteria. We de novo sequence the genomes or transcriptomes of 12 lichen algal symbiont (LAS) and closely related non-symbiotic algae (NSA) to improve the genomic coverage of Chlorophyte algae. We then perform ancestral state reconstruction and comparative phylogenomics. We identify at least three independent gains of the ability to engage in the lichen symbiosis, one in Trebouxiophyceae and two in Ulvophyceae, confirming the convergent evolution of the lichen symbioses. A carbohydrate-active enzyme from the glycoside hydrolase 8 (GH8) family was identified as a top candidate for the molecular-mechanism underlying lichen symbiosis in Trebouxiophyceae. This GH8 was acquired in lichenizing Trebouxiophyceae by horizontal gene transfer, concomitantly with the ability to associate with lichens fungal symbionts (LFS) and is able to degrade polysaccharides found in the cell wall of LFS. These findings indicate that a combination of gene family expansion and horizontal gene transfer provided the basis for lichenization to evolve in chlorophyte algae.
Asunto(s)
Chlorophyta , Líquenes , Filogenia , Simbiosis , Líquenes/genética , Líquenes/microbiología , Simbiosis/genética , Chlorophyta/genética , Transferencia de Gen Horizontal , Evolución Molecular , Evolución Biológica , Transcriptoma , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , GenómicaRESUMEN
Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade various polysaccharides, such as cellulose. Despite extensive research on this class of enzymes, the role played by their C-terminal regions predicted to be intrinsically disordered (dCTR) has been overlooked. Here, we investigated the function of the dCTR of an LPMO, called CoAA9A, up-regulated during plant infection by Colletotrichum orbiculare, the causative agent of anthracnose. After recombinant production of the full-length protein, we found that the dCTR mediates CoAA9A dimerization in vitro, via a disulfide bridge, a hitherto-never-reported property that positively affects both binding and activity on cellulose. Using SAXS experiments, we show that the homodimer is in an extended conformation. In vivo, we demonstrate that gene deletion impairs formation of the infection-specialized cell called appressorium and delays penetration of the plant. Using immunochemistry, we show that the protein is a dimer not only in vitro but also in vivo when secreted by the appressorium. As these peculiar LPMOs are also found in other plant pathogens, our findings open up broad avenues for crop protection.