An oomycete NLP cytolysin forms transient small pores in lipid membranes

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.Peer reviewe

Effect of mutations in the coat protein of potato virus Y on biochemical and biophysical properties as well as structure of virus-like particles

Virusom podobni delci (angl. virus-like particles, VLPs) so strukturno podobni virusom, iz katerih so pridobljeni, a za razliko od izvornih virusov nimajo prisotnega virusnega genetskega materiala. Lastnosti VLP-jev, ki doprinašajo k vedno večjemu pomenu VLP-jev na področju nanobiotehnoloških aplikacij, so simetrična ureditev plaščnih proteinov, enostavne metode izolacije, dovzetnost za genske in proteinske modifikacije in varna uporaba v bioloških sistemih. Virus Y krompirja (angl. potato virus Y, PVY) tvori do 740 nm dolge in 13 nm široke virione, plaščni proteini pa se ob izražanju v bakterijskem ekspresijskem sistemu ter odsotnosti virusne RNA samosestavijo v do 3 µm dolge in 13 nm široke nitaste fleksibilne delce. V raziskovalni skupini, kjer sem opravljala magistrsko nalogo, so določili tridimenzionalno strukturo virusa PVY in ustreznega VLP-ja s pomočjo krio-elektronske mikroskopije pri ločljivosti blizu atomske. V primeru VLP-ja so ugotovili, da ena enota plaščnega proteina (angl. coat protein, CP) sestavlja osemčlenske obroče, ki se med seboj sestavljajo v dolge nitke. Namen mojega magistrskega dela je bil pripraviti take rekombinantne oblike CP-ja, ki bi dale nastalim VLP-jem posebne lastnostni za nadaljno uporabo v različne bionanotehnološke namene. Na podlagi znane strukture VLP-ja smo pripravili mutante CP-ja tako, da smo določene aminokislinske ostanke, izpostavljene na površini VLP nitke, zamenjali s cisteinskim ostankom. Po izražanju v Escherichia coli smo samosestavljanje mutiranih CP-jev v VLP-je potrdili s presevnim elektronskim mikroskopom. Da uvedba mutacij ni vplivala na sekundarno strukturo plaščnega proteina smo pokazali s cirkularnim dikroizmom. O termični stabilnosti smo sklepali na podlagi temperatur denaturacije, ki smo jih določili z metodo dinamične diferenčne fluorimetrije. Izkazalo se je, da je vpliv sprememb pH in ionske jakosti na termično stabilnost mutantov primerljiv z vplivom na stabilnost PVY VLP-jev. Prisotnostnost uvedenih reaktivnih tiolnih funkcionalnih skupin na površini VLP-jev smo pokazali z vezavo fluorescenčnega barvila Alexa Fluor 488, ki specifično prepozna tiolne funkcionalne skupine. Termično tabilni ter biokemijsko in biofizikalno okarakterizirani PVY VLP-ji s površinsko prisotnimi reaktivnimi tiolnimi funkcionalnim skupinami so prvi PVY VLP-ji s tovrstno modifikacijo in tako odpirajo nove možnosti za modificiranje fleksibilnih nitastih delcev.Virus-like particles (VLPs) are similar to their native viruses, but they are lacking viral genetic material. Highly symmetrical coat protein organisation, simple production, easy genetic and protein modifications as well as safe usage in biological systems are some of the main reasons why VLPs are gaining importance in nanobiotehnological field. Potato virus Y (PVY) virions are 740 nm long and 13 nm wide. When coat proteins are overexpressed in bacterial system, they self-assemble into up to 3 µm long and 13 nm wide flexible filamentous VLPs. Experimental part of my master thesis was done in a research group that recently determined three-dimensional structure of PVY virions by crio-electron microscopy at near atomic resolution. In the case of VLP they found out that CPs are organised in octamer rings that stack together and form long flexible particles. The goal of my thesis was to prepare recombinant CPs that would introduce special characteristic to VLPs and make them suitable for different applications. Depending on the known structure of VLP we prepared mutants of CP in the way that we introduced novel cysteine on the outer VLP’s surface. The self-assembly of VLPs after overexpression of CP mutants in Escherichia coli was confirmed by transmission electron microscope. For those mutants we also measured circular dichroism and confirm that substitutions did not disturb CP’s secondary structure. Comparison of melting temperatures, determined by differential scanning fluorimetry, showed that mutants tolerate harsher conditions referring to extreme pH and higher concentrations of salt. The introduction of novel cysteines on the VLP’s outer surface was confirmed by binding of fluorescent dye Alexa Fluor 488, that preferentially react only with thiol functional groups. During this thesis we produced and characterised, for the first time, stable PVY VLPs with novel reactive functional groups on their outer surface and thus open new possibilities for further modification of flexible filamentous particles

Biological nanopores : engineering on demand

Nanopore-based sensing is a powerful technique for the detection of diverse organic and inorganic molecules, long-read sequencing of nucleic acids, and single-molecule analyses of enzymatic reactions. Selected from natural sources, protein-based nanopores enable rapid, label-free detection of analytes. Furthermore, these proteins are easy to produce, form pores with defined sizes, and can be easily manipulated with standard molecular biology techniques. The range of possible analytes can be extended by using externally added adapter molecules. Here, we provide an overview of current nanopore applications with a focus on engineering strategies and solutions

An oomycete NLP cytolysin forms transient small pores in lipid membranes

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.Peer reviewe