Biology subjects

Sprakel, J.

Publications and source records attributed to Sprakel, J..

8 recordsLinked to original sources

bioRxiv2024

H+ drives ultra-fast root-to-root responses to wounding

The plant-to-plant communication of damage is vital for plants to mount pre-emptive defensive responses in the face of threats. A variety of threats to the well-being of plants are found below ground; yet how plant roots activate inter-plant communication is largely unclear. Here we demonstrate that a wounded root rapidly releases protons (H+), that travel faster than any other "known" soluble biochemical signal due to a specialised diffusion mechanism. Within seconds after damage, cells in neighbouring unwounded roots sense the acidification and activate tissue-specific Ca2+ damage signalling. In turn, this triggers a differential growth response allowing the unwounded root to avoid the site of a potential threat. Our results reveal a non-canonical rapid response mechanism for inter-plant communication based on ultrafast proton diffusion.

plant biology↗

bioRxiv2024

CarboTag: a modular approach for live and functional imaging of plant cell walls

Plant cells are contained inside a rigid network of cell walls. Cell walls are highly dynamic structures that act both as a structural material and as a hub for a wide range of signaling processes. Despite its crucial role in all aspects of the plant life cycle, live dynamical imaging of the cell wall and its functional properties has remained challenging. Here, we introduce CarboTag, a modular toolbox for live functional imaging of plant walls. CarboTag relies on a small molecular motif, a pyridine boronic acid, that targets its cargo to the cell wall, is non-toxic and ensures rapid tissue permeation. We designed a suite of cell wall imaging probes based on CarboTag in any desired color for multiplexing. Moreover, we created new functional reporters for live quantitative imaging of key cell wall features: network porosity, cell wall pH and the presence of reactive oxygen species. CarboTag opens the way to dynamical and quantitative mapping of cell wall responses at subcellular resolution.

plant biology↗

bioRxiv2024

ATG8ylation of vacuolar membrane protects plants against cell wall damage

Vacuoles are essential for cellular metabolism, growth, and the maintenance of internal turgor pressure. They sequester lytic enzymes, ions, and secondary metabolites that, if leaked into the cytosol, could lead to cell death. Despite their pivotal roles, quality control pathways that safeguard vacuolar integrity remained elusive in plants. Here, we discovered a conserved vacuolar quality control (VQC) pathway that is activated upon cell wall damage in a turgor pressure dependent manner. Cell wall perturbations induce a distinct modification - ATG8ylation - on the vacuolar membrane (tonoplast) that is regulated by the V-ATPase and ATG8 conjugation machinery. Genetic disruption of tonoplast ATG8ylation impairs vacuolar integrity, leading to cell death. Together, our findings reveal a homeostatic pathway that preserves vacuolar integrity upon cell wall damage.

plant biology↗

bioRxiv2023

Plant infection by the necrotrophic fungus Botrytis requires actin-dependent generation of high invasive turgor pressure

The devastating pathogen Botrytis cinerea infects a broad spectrum of host plants, causing great socio-economic losses. The necrotrophic fungus rapidly kills plant cells, nourishing their walls and cellular contents. To this end, necrotrophs secretes a cocktail of cell wall degrading enzymes, phytotoxic proteins and metabolites. Additionally, many fungi produce specialized invasion organs that generate high invasive pressures to force their way into the plant cell. However, for most necrotrophs, including Botrytis, the biomechanics of penetration and its contribution to virulence are poorly understood. Here we use a combination of quantitative micromechanical imaging and CRISPR-Cas guided mutagenesis to show that Botrytis uses substantial invasive pressure, in combination with strong surface adherence, for penetration. We found that the fungus establishes a unique mechanical geometry of penetration that develops over time during penetration events, and which is actin cytoskeleton dependent. Furthermore, interference of force generation by blocking actin polymerization was found to decrease Botrytis virulence, indicating that also for necrotrophs, mechanical pressure is important in host colonization. Our results demonstrate for the first time mechanistically how a necrotrophic fungus such as Botrytis employs this "brute force" approach, in addition to the secretion of lytic proteins and phytotoxic metabolites, to overcome plant host resistance.

biophysics↗

bioRxiv2023

An elastic proteinaceous envelope encapsulates the early Arabidopsis embryo

Plant external surfaces are often covered by barriers that control the exchange of molecules, protect from pathogens, and offer mechanical integrity. A key question is when and how such surface barriers are generated. Post-embryonic surfaces have well-studied barriers, including the cuticle, and late Arabidopsis embryo was shown to be protected by an endosperm-derived sheath deposited onto a primordial cuticle. Here we show that both cuticle and sheath are preceded by another structure during the earliest stages of embryogenesis. This structure, which we named the embryonic envelope, is tightly wrapped around the embryonic surface but can be physically detached by cell wall digestion. We show that this structure is composed primarily of Extensin and Arabinogalactan O-glycoproteins and lipids, which appear to form a dense and elastic crosslinked embryonic envelope. The envelope forms in cuticle-deficient mutants and in a mutant that lacks endosperm. This embryo-derived envelope is therefore distinct from previously described cuticle and sheath structures. We propose that it acts as an expandable diffusion barrier, as well as a means to mechanically confine the embryo to maintain its tensegrity during early embryogenesis. Summary statementThe early Arabidopsis embryo is surrounded by a proteinaceous envelope that is distinct from the cuticle or embryo sheath

plant biology↗

bioRxiv2023

Rapid Molecular Mechanotyping with Microfluidic Force Spectroscopy

Molecular mechanotyping, the quantification of changes in the stability of supramolecular interactions and chemical bonds under the action of mechanical forces, is an essential tool in the field of mechanochemistry. This is conventionally done in single-molecule force-spectroscopy (smFS) assays, for example with optical tweezers or Atomic Force Microscopy. While these techniques provide detailed mechanochemical insights, they are time-consuming, technically demanding and expensive; as a result, high-throughput screening of the mechanochemical properties of molecules of interest is challenging. To resolve this, we present a rapid, simple and low-cost mechanotyping assay: microfluidic force spectroscopy ({micro}FFS), which probes force-dependent bond stability by measuring the detachment of microparticles, bound to microfluidic channels by the interaction of interest, under hydrodynamic forcing. As this allows the simultaneous observation of hundreds of microparticles, we obtain a quantitative mechanotype in a single measurement, using readily available equipment. We validate our method by studying the stability of DNA duplexes, previously characterized through smFS. We further show that we can quantitatively describe the experimental data with simulations, which allows us to link the {micro}FFS data to single-bond mechanochemical properties. This opens the way to use ({micro}FFS) as a rapid molecular mechanotyping tool.

biophysics↗

bioRxiv2022

A RAF-like kinase mediates a deeply conserved, ultra-rapid auxin response

The plant signaling molecule auxin triggers both fast and slow cellular responses across the plant kingdom, including both land plants and algae. A nuclear response pathway mediates auxin-dependent gene expression, and controls a range of growth and developmental processes in land plants. It is unknown what mechanisms underlie both the physiological responses occurring within seconds, and the responses in algae, that lack the nuclear auxin response pathway. We discovered an ultra-fast proteome-wide phosphorylation response to auxin across 5 land plant and algal species, converging on a core group of shared target proteins. We find conserved rapid physiological responses to auxin in the same species and identified a RAF-like protein kinase as a central mediator of auxin-triggered phosphorylation across species. Genetic analysis allowed to connect this kinase to both auxin-triggered protein phosphorylation and a rapid cellular response, thus identifying an ancient mechanism for fast auxin responses in the green lineage.

plant biology↗

bioRxiv2022

Direct measurement of appressorium turgor using a molecular mechanosensor in the rice blast fungus Magnaporthe oryzae

Many plant pathogenic fungi forcibly enter their hosts to cause disease. The rice blast fungus Magnaporthe oryzae, for example, infects plants using a specialised infection cell called an appressorium, which generates enormous turgor to drive a rigid penetration peg through the rice leaf cuticle. While these vast internal pressures are a critical weapon in fungal host penetration, they have remained very challenging to probe directly during host invasion, leaving our understanding of these extreme cellular mechanics incomplete. Here, we combine Fluorescence Lifetime Imaging (FLIM) with a membrane-targeting molecular mechanoprobe to quantify changes in membrane tension as a direct proxy for appressorial turgor in M. oryzae. We report that mature melanin-pigmented M. oryzae appressoria display a heterogeneous low fluorescence lifetime and high membrane tension, consistent with enormous turgor. These extreme pressures lead to large-scale spatial heterogeneities in membrane mechanics, much greater than observed in any other cell type previously, highlighting the extreme mechanics of turgor-driven appressorium-mediated plant infection. By contrast, appressoria of non-pathogenic melanin-deficient mutants, alb1 and buf1, or immature non-melanised appressoria, exhibit high fluorescence lifetime, consistent with low membrane tension and turgor, that remain spatially homogeneous. To evaluate the method, we investigated turgor dynamics in a range of mutants impaired in appressorium function. We show that the turgor sensor kinase mutant{Delta} sln1, recently proposed to generate excess appressorium turgor, displayed a significantly higher membrane tension compared to an isogenic wild type M. oryzae strain. This non-invasive, live cell imaging technique allows direct quantification and visualization of the enormous turgor pressures deployed during pathogen infection.

cell biology↗