Biology subjects

Denninger, P.

Publications and source records attributed to Denninger, P..

5 recordsLinked to original sources

bioRxiv2026

A GreenGate-compatible vector set for efficient protein purification from E. coli

Recombinant protein purification from E. coli frequently requires screening various affinity tags and variations to optimize yield and purity. However, classical cloning methods are limited in throughput and modularity, which is circumvented by GoldenGate cloning. While GreenGate cloning, a GoldenGate variant, is widely used in plant research, it lacks compatibility with E. coli expression vectors. Here, we introduce a comprehensive, GreenGate-compatible vector toolkit for efficient and versatile assembly of three modules, for N- and C-terminal tagging of a protein of interest into an IPTG-inducible E. coli expression vector. To allow versatility, this toolkit contains diverse affinity tags, with or without HRV3C protease cleavage sites. Moreover, we included plasmids for the homemade low-cost production of this protease. This GreenGate-compatible toolkit allows efficient combinations of different tags and eliminates the need for re-cloning modules between plant and bacterial systems, streamlining the workflow for recombinant protein production, especially in plant research.

Plant Biology↗

bioRxiv2024

The Pollen Tube Penetrates the Synergid Cell by Formation of a Peritubular Membrane

In flowering plants, successful reproduction relies on an exchange of signals between synergids and pollen tubes (PTs), mediating the invasion of a synergid by the PT, which then ruptures and releases two sperm cells to effect double fertilization. However, how exactly the PT invades the receptive synergid is unknown as the spatial relationship between these two cells is unclear. To better understand this process we performed 3D live imaging of PT reception in Arabidopsis thaliana. Upon arrival at the filiform apparatus (FA), a region rich in membrane folds at the micropylar pole of the synergids, the PT gradually deforms the FA before it rapidly grows into the receptive synergid. Upon penetration, the membrane of the receptive synergid invaginates and envelopes the PT. We termed this newly discovered structure the peri-tubular membrane (PRM). We show that, in feronia mutants disrupting PT reception, the PT still enters the receptive synergid, forming a normal PRM. This results in extensive invagination of the synergid membrane without sperm release. We show that PRM formation is associated with a cytosolic calcium ([Ca2+]cyt) spike of high amplitude in the PT and flooding of [Ca2+]cyt in the synergids. In PTs lacking AUTOINHIBITED Ca2+ ATPASE9 activity, PTs have lower amplitude [Ca2+]cyt spiking and the PTs frequently fail to penetrate the synergid. Our findings suggest that synergid penetration and the non-cell autonomous control of PT rupture are distinct regulated processes required for fertilization in flowering plants.

plant biology↗

bioRxiv2024

Polarised subcellular activation of ROPs by specific ROPGEFs drives pollen germination in Arabidopsis thaliana

During plant fertilisation, excess male gametes compete for a limited number of female gametes. The dormant male gametophyte, encapsulated in the pollen grain, consists of two sperm cells enclosed in a vegetative cell. After reaching the stigma of a compatible flower, quick and efficient germination of the vegetative cell to a tip-growing pollen tube is crucial to ensure fertilisation success. RHO OF PLANTS (ROP) signalling and their activating ROP GUANINE NUCLEOTIDE EXCHANGE FACTORS (ROPGEFs) are essential for initiating polar growth processes in multiple cell types. However, which ROPGEFs activate pollen germination is unknown. We investigated the role of ROPGEFs in initiating pollen germination and the required cell polarity establishment. Of the five pollen-expressed ROPGEFs, we found that GEF8, GEF9, and GEF12 are required for pollen germination and male fertilisation success, as gef8;gef9;gef12 triple mutants showed almost complete loss of pollen germination in vitro and had a reduced allele transmission rate. Live cell imaging and spatiotemporal analysis of subcellular protein distribution showed that GEF8 and GEF9, but not GEF12, displayed transient polar protein accumulations at the future site of pollen germination minutes before pollen germination, demonstrating specific roles for GEF8 and GEF9 during the initiation of pollen germination. Furthermore, this novel GEF accumulation appears in a biphasic temporal manner and can shift its location. We showed that the C-terminal domain of GEF8 and GEF9 confers this protein accumulation and demonstrated that GEFs locally activate ROPs and alter Ca2+ signalling, which is required for pollen tube germination. We demonstrated that GEFs do not act redundantly during pollen germination and described for the first time a polar domain with spatiotemporal flexibility, which is crucial for the de novo establishment of a polar growth domain within a cell and, thus, for pollen function and fertilisation success.

plant biology↗

bioRxiv2021

Nanodomain-mediated lateral sorting drives polarization of the small GTPase ROP2 in the plasma membrane of root hair cells

Formation of root hairs involves the targeted recruitment of the cellular growth machinery to the root hair initiation domain (RHID), a specialized site at the plasma membrane (PM) of trichoblast cells. Early determinants in RHID establishment are small GTPases of the Rho-of-plants (ROP) protein family, which are required for polarization of downstream effectors, membrane modification and targeted secretion during tip growth. It remains, however, not fully understood how ROP GTPases themselves are polarized. To investigate the mechanism underlying ROP2 recruitment, we employed Variable Angle Epifluorescence Microscopy (VAEM) and exploited mCitrine fluorophore blinking for single molecule localization, particle tracking and super-resolved imaging of the trichoblast plasma membrane. We observed the association of mCit-ROP2 within distinct membrane nanodomains, whose polar occurrence at the RHID was dependent on the presence of the RopGEF GEF3, and found a gradual, localized decrease of mCit-ROP2 protein mobility that preceded polarization. We provide evidence for a step-wise model of ROP2 polarization that involves (i) an initial non-polar recruitment to the plasma membrane via interactions with anionic phospholipids, (ii) ROP2 assembly into membrane nanodomains independent of nucleotide-binding state and, sub-sequently, (iii) lateral sorting into the RHID, driven by GEF3-mediated localized reduction of ROP2 mobility.

cell biology↗

bioRxiv2019

Cytoskeleton dynamics control early events of lateral root initiation in Arabidopsis

How plant cells re-establish differential growth to initiate organs is poorly understood. Morphogenesis of lateral roots relies on the tightly controlled radial expansion and asymmetric division of founder cells. The cellular mechanisms that license and ensure these features are unknown. Here, we quantitatively analyse F-actin and microtubule dynamics during LR initiation. Using mutants, pharmacological and tissue-specific genetic perturbations, we show that dynamic reorganisation of both microtubule and F-actin networks is required for the asymmetric expansion of the founder cells. This cytoskeleton remodelling intertwine with auxin signalling in the pericycle and endodermis in order for founder cells to acquire a basic polarity required for initiating LR development. Our results reveal the conservation of cell remodelling and polarisation strategies between the Arabidopsis zygote and lateral root founder cells. We propose that coordinated, auxin-driven reorganisation of the cytoskeleton licenses asymmetric cell growth and divisions during embryonic and post-embryonic organogenesis. HIGHLIGHTSO_LIFailure for lateral root founder cells to undergo asymmetric radial expansion before division, leads to aberrant organ formation. C_LIO_LICortical microtubules arrays reorganise to facilitate this asymmetric expansion and F-actin the asymmetric division. C_LIO_LICytoskeletal reorganisation depends on auxin signalling. C_LIO_LINew genetic tools allow to perturb microtubules or actin in an inducible and cell-type specific manner. C_LI

plant biology↗