High efficiency transient and stable transformation by optimized DNA microinjection into Nicotiana tabacum protoplasts

An efficient system has been established that allows well controlled DNA microinjection into tobacco (Nicotiana tabacum) mesophyll protoplasts with partially regenerated cell walls and subsequent analysis of transient as well as stable expression of injected reporter genes in particular targeted cells or derived clones. The system represents an effective tool to study parameters important for the successful transformation of plant cells by microinjection and other techniques. Protoplasts were immobilized in a very thin layer of medium solidified with agarose or alginate. DNA microinjection was routinely monitored by coinjecting FITC-dextran and aimed at the cytoplasm of target cells. The injection procedure was optimized for efficient delivery of injection solution into this compartment. Cells were found to be at the optimal stage for microinjection about 24 h after immobilization in solid medium. Embedded cells could be kept at this stage for up to 4 d by incubating them at 4 °C in the dark. Within 1 h successful delivery of injection, solution was routinely possible into 20-40 cells. Following cytoplasmic coinjection of FITC-dextran and pSHI913, a plasmid containing the neo (neomycin phosphotransferase II) gene, stably transformed, paromomycin-resistant clones could be recovered through selection. Transgenic tobacco lines have been established from such clones. Injection solutions containing pSHI913 at a concentration of either 50 μg ml−1 or 1 mg ml−1 have been tested. With 1 mg ml−1 plasmid DNA the percentage of resistant clones per successfully injected cell was determined to be about 3.5 times higher. Incubation of embedded protoplasts at 4°C before microinjection was found to reduce the percentage of resistant clones obtained per injected cell Protoplasts were immobilized above a grid pattern and the location of injected cells was recorded by Polaroid photography. The fate of particular targeted cells could be observed. Isolation and individual culture of clones derived from injected cells was possible. Following cytoplasmic coinjection of FITC-dextran and 1 mg ml−1 plasmid DNA on average about 20% of the targeted cells developed into microcalli and roughly 50% of these calli were stably transformed. Transient expression of the firefly luciferase gene (Luc) was nondestructively analysed 24 h after injection of pAMLuc. Approximately 50% of the injected cells that were alive at this time point expressed the Luc gene transiently. Apparently, stable integration of the injected genes occurred in essentially all transiently expressing cells that developed into clone

Analysis of the Localization of Fluorescent PpROP1 and PpROP-GEF4 Fusion Proteins in Moss Protonemata Based on Genomic “Knock-In” and Estradiol-Titratable Expression

Tip growth of pollen tubes, root hairs, and apical cells of moss protonemata is controlled by ROP (Rho of plants) GTPases, which were shown to accumulate at the apical plasma membrane of these cells. However, most ROP localization patterns reported in the literature are based on fluorescent protein tagging and need to be interpreted with caution, as ROP fusion proteins were generally overexpressed at undefined levels, in many cases without assessing effects on tip growth. ROP-GEFs, important regulators of ROP activity, were also described to accumulate at the apical plasma membrane during tip growth. However, to date only the localization of fluorescent ROP-GEF fusion proteins strongly overexpressed using highly active promoters have been investigated. Here, the intracellular distributions of fluorescent PpROP1 and PpROP-GEF4 fusion proteins expressed at essentially endogenous levels in apical cells of Physcomitrella patens “knock-in” protonemata were analyzed. Whereas PpROP-GEF4 was found to associate with a small apical plasma membrane domain, PpROP1 expression was below the detection limit. Estradiol-titratable expression of a fluorescent PpROP1 fusion protein at the lowest detectable level, at which plant development was only marginally affected, was therefore employed to show that PpROP1 also accumulates at the apical plasma membrane, although within a substantially larger domain. Interestingly, RNA-Seq data indicated that the majority of all genes active in protonemata are expressed at lower levels than PpROP1, suggesting that estradiol-titratable expression may represent an important alternative to “knock-in” based analysis of the intracellular distribution of fluorescent fusion proteins in protonemal cells

Physcomitrium patens PpRIC, an ancestral CRIB-domain ROP effector, inhibits auxin-induced differentiation of apical initial cells

RHO guanosine triphosphatases are important eukaryotic regulators of cell differentiation and behavior. Plant ROP (RHO of plant) family members activate specific, incompletely characterized downstream signaling. The structurally simple land plant Physcomitrium patens is missing homologs of key animal and flowering plant RHO effectors but contains a single CRIB (CDC42/RAC interactive binding)-domain -contain-ing RIC (ROP-interacting CRIB-containing) protein (PpRIC). Protonemal P. patens filaments elongate based on regular division and PpROP-dependent tip growth of apical initial cells, which upon stimulation by the hor-mone auxin differentiate caulonemal characteristics. PpRIC interacts with active PpROP1, co-localizes with this protein at the plasma membrane at the tip of apical initial cells, and accumulates in the nucleus. Remark-ably, PpRIC is not required for tip growth but is targeted to the nucleus to block caulonema differentiation downstream of auxin-controlled gene expression. These observations establish functions of PpRIC in medi-ating crosstalk between ROP and auxin signaling, which contributes to the maintenance of apical initial cell identity

Spatial control of Rho (Rac-Rop) signaling in tip-growing plant cells

Spatially restricted signaling by Rho GTPases is essential for the polarization of eukaryotic cells, which is required for the morphogenesis, mobility and division of single cells, and for the development of multicellular organisms. Rac-Rop GTPases, which constitute a plant-specific Rho GTPase subfamily, accumulate at the apical plasma membrane of pollen tubes and root hairs, where they control rapid polar cell expansion by a process known as tip growth. Here, recent insights into the spatial control of Rac-Rop-dependent signaling in tip-growing plant cells by regulatory proteins (i.e. Rho GTPase-activating proteins, Rho guanine nucleotide dissociation inhibitors, Rho guanine nucleotide-exchange factors and phosphoinositide-specific phospholipase C) and lipids [phosphatidylinositol (4,5)-bisphosphate and diacyl glycerol] are summarized. A model is presented, which integrates the current knowledge concerning the molecular mechanisms that maintain the polarization of Rho signaling in plant cells

Tobacco RhoGTPase ACTIVATING PROTEIN1 spatially restricts signaling of RAC/Rop to the apex of pollen tubes

Regulation by Rho-type small GTPases, such as RAC5, is important for the maintenance of polarity in tobacco (Nicotiana tabacum) pollen tubes. We previously showed that RhoGDI2 is necessary for RAC5 localization. Here, we describe the GTPase activating protein RhoGAP1 that controls the area of RAC5 activity. RhoGAP1 N-terminal and CRIB (for Cdc42/Rac-interactive binding) domains are both necessary for targeting yellow fluorescent protein–RhoGAP1 fusions to the plasma membrane close to, but not in, pollen tube apices. We propose that this localization restricts apical Rho-type GTPase activity from spreading toward the flanks, which ensures the maintenance of RAC signaling at the apex. The CRIB domain is not required but enhances in vitro RhoGAP1 activity toward the pollen tube–specific-RAC5. A mutation reducing GAP activity of RhoGAP1 leads to ballooning pollen tubes resembling those overexpressing RAC5. To ascertain the specific targeting mechanism of RhoGAP1, we isolated a 14-3-3 protein interacting with RhoGAP1. When overexpressed with RhoGAP1, it counteracts the growth-retarding effect of RhoGAP1 overexpression and attenuates RhoGAP1 membrane localization but, overexpressed alone, induces only small architectural changes. We propose that inactivation of RAC5 by the subapically localized RhoGAP1, together with dynamic relocalization of inactivated RAC5 from flanks to tip by RhoGDI2, leads to spatial restriction of RAC5 to pollen tube apices, thereby sustaining polar growth

Direct Comparison of the Performance of Commonly Employed In Vivo F-actin Markers (Lifeact-YFP, YFP-mTn and YFP-FABD2) in Tobacco Pollen Tubes

In vivo markers for F-actin organization and dynamics are extensively used to investigate cellular functions of the actin cytoskeleton, which are essential for plant development and pathogen defense. The most widely employed markers are GFP variants fused to F-actin binding domains of mouse talin (GFP-mTn), Arabidopsis fimbrin1 (GFP-FABD2) or yeast Abp140 (Lifeact-GFP). Although numerous reports describing applications of one, or occasionally more, of these markers, are available in the literature, a direct quantitative comparison of the performance of all three markers at different expression levels has been missing. Here, we analyze F-actin organization and growth rate displayed by tobacco pollen tubes expressing YFP-mTn, YFP-FABD2 or Lifeact-YFP at different levels. Results obtained establish that: (1) all markers strongly affect F-actin organization and cell expansion at high expression levels, (2) YFP-mTn and Lifeact-YFP non-invasively label the same F-actin structures (longitudinally oriented filaments in the shank, a subapical fringe) at low expression levels, (3) Lifeact-YFP displays a somewhat lower potential to affect F-actin organization and cell expansion than YFP-mTn, and (4) YFP-FABD2 generally fails to label F-actin structures at the pollen tube tip and affects F-actin organization as well as cell expansion already at lowest expression levels. As pointed out in the discussion, these observations (1) are also meaningful for F-actin labeling in other cell types, which generally respond less sensitively to F-actin perturbation than pollen tubes, (2) help selecting suitable markers for future F-actin labeling experiments, and (3) support the assessment of a substantial amount of published data resulting from such experiments

TETRASPANINs in Plants

Tetraspanins are small transmembrane proteins that laterally associate with each other and cluster with numerous partner proteins as well as lipids. These interactions result in the formation of a distinct class of membrane domains, the tetraspanin-enriched microdomains (TEMs), which influence numerous cellular processes such as cell adhesion and fusion, intracellular membrane trafficking, signaling, morphogenesis, motility as well as interaction with pathogens and cancer development. The majority of information available about tetraspanins is based on studies using animal models or cell lines, but tetraspanins are also present in fungi and plants. Recent studies indicate that tetraspanins have important functions in plant development, reproduction and stress responses. Here we provide a brief summary of the current state of tetraspanin research in plants