A ubiquitin‐10 promoter‐based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies

Fluorescent tagging of proteins and confocal imaging techniques have become methods of choice in analysing the distributions and dynamic characteristics of proteins at the subcellular level. In common use are a number of strategies for transient expression that greatly reduce the preparation time in advance of imaging, but their applications are limited in success outside a few tractable species and tissues. We previously developed a simple method to transiently express fluorescently‐tagged proteins in Arabidopsis root epidermis and root hairs. We describe here a set of Gateway‐compatable vectors with fluorescent tags incorporating the ubiqutin‐10 gene promoter (P_UBQ10) of Arabidopsis that gives prolonged expression of the fluorescently‐tagged proteins, both in tobacco and Arabidopsis tissues, after transient transformation, and is equally useful in generating stably transformed lines. As a proof of principle, we carried out transformations with fluorescent markers for the integral plasma membrane protein SYP121, a member of the SNARE family of vesicle‐trafficking proteins, and for DHAR1, a cytosolic protein that facilitates the scavenging of reactive oxygen species. We also carried out transformations with SYP121 and its interacting partner, the KC1 K⁺ channel, to demonstrate the utility of the methods in bimolecular fluorescence complementation (BiFC). Transient transformations of Arabidopsis using Agrobacterium co‐cultivation methods yielded expression in all epidermal cells, including root hairs and guard cells. Comparative studies showed that the P_UBQ10 promoter gives similar levels of expression to that driven by the native SYP121 promoter, faithfully reproducing the characteristics of protein distributions at the subcellular level. Unlike the 35S‐driven construct, expression under the P_UBQ10 promoter remained elevated for periods in excess of 2 weeks after transient transformation. This toolbox of vectors and fluorescent tags promises significant advantages for the study of membrane dynamics and cellular development, as well as events associated with environmental stimuli in guard cells and nutrient acquisition in roots.     

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.     

Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells

Transient expression of fluorescent fusion proteins in plant cells has dramatically facilitated our study of newly identified genes and proteins. This protocol details an in vivo transient expression system to study the subcellular localization and dynamic associations of plant proteins using protoplasts freshly prepared from Arabidopsis or tobacco BY-2 suspension cultured cells. The method relies on the transformation of DNA constructs into protoplasts via electroporation. The whole protocol is comprised of three major stages: protoplast generation and purification, transformation of DNA into protoplasts via electroporation and incubation of protoplasts for protein analysis. Similar to stably transformed cell lines, transformed protoplasts are compatible with protein localization studies, pharmaceutical drug treatment and western blot analysis. This protocol can be completed within 11-24 h from protoplast production to protein detection.     

New guidelines for fluorophore application in peroxisome targeting analyses in transient plant expression systems

Peroxisome research has been revolutionized by proteome studies combined with in vivo subcellular targeting analyses. Yellow and cyan fluorescent protein(YFP and CFP) are the classical fluorophores of plant peroxisome research. In the new transient expression system of Arabidopsis seedlings co-cultivated with Agrobacterium we detected the YFP fusion of one candidate protein in peroxisomes, but only upon co-transformation with the peroxisome marker, CFP-PTS_1. The data suggested that the YFP fusion was directed to peroxisomes due to its weak heterodimerization ability with CFP-PTS_1,allowing piggy-back import into peroxisomes. Indeed, if co-expressed with monomeric Cerulean-PTS_1(mCer-PTS_1),the YFP fusion was no longer matrix localized. We systematically investigated the occurrence and extent of dimerization-based piggy-back import for different fluorophore combinations in five major transient plant expression systems. In Arabidopsis seedlings and tobacco leaves both untagged YFP and monomeric Venus were imported into peroxisomes if co-expressed with CFP-PTS_1 but not with mCer-PTS_1. By contrast, piggy-back import of cytosolic proteins was not observed in Arabidopsis and tobacco protoplasts or in onion epidermal cells for any fluorophore combination at any time point. Based on these important results we formulate new guidelines for fluorophore usage and experimental design to guarantee reliable identification of novel plant peroxisomal proteins.     

Characterization of stress‐responsive lncRNAs in Arabidopsis thaliana by integrating expression,epigenetic and structural features

Agrobacterium genetically transforms plants by transferring and integrating T‐(transferred) DNA into the host genome. This process requires both Agrobacterium and host proteins. VirE2 interacting protein 1 (VIP1), an Arabidopsis bZIP protein, has been suggested to mediate transformation through interaction with and targeting of VirE2 to nuclei. We examined the susceptibility of Arabidopsis vip1 mutant and VIP1 overexpressing plants to transformation by numerous Agrobacterium strains. In no instance could we detect altered transformation susceptibility. We also used confocal microscopy to examine the subcellular localization of Venus‐tagged VirE2 or Venus‐tagged VIP1, in the presence or absence of the other untagged protein, in different plant cell systems. We found that VIP1–Venus localized in both the cytoplasm and the nucleus of Arabidopsis roots, agroinfiltrated Nicotiana benthamiana leaves, Arabidopsis mesophyll protoplasts and tobacco BY‐2 protoplasts, regardless of whether VirE2 was co‐expressed. VirE2 localized exclusively to the cytoplasm of tobacco and Arabidopsis protoplasts, whether in the absence or presence of VIP1 overexpression. In transgenic Arabidopsis plants and agroinfiltrated N. benthamina leaves we could occasionally detect small aggregates of the Venus signal in nuclei, but these were likely to be imagining artifacts. The vast majority of VirE2 remained in the cytoplasm. We conclude that VIP1 is not important for Agrobacterium‐mediated transformation or VirE2 subcellular localization.     

A bicistronic, Ubiquitin-10 promoter-based vector cassette for transient transformation and functional analysis of membrane transport demonstrates the utility of quantitative voltage clamp studies on intact Arabidopsis root epidermis

To date the use of fluorescent reporter constructs in analysing membrane transport has been limited primarily to cell lines expressing stably either the tagged transporter protein(s) or markers to identify lineages of interest. Strategies for transient expression have yet to be exploited in transport analysis, despite their wide application in cellular imaging studies. Here we describe a Gateway-compatible, bicistronic vector, incorporating the constitutive Ubiqutin-10 gene promoter of Arabidopsis that gives prolonged expression after transient transformation and enables fluorescence marking of cells without a fusion construct. We show that Arabidopsis root epidermal cells are readily transformed by co-cultivation with Agrobacterium and are tractable for quantitative electrophysiological analysis. As a proof of principle, we transiently transformed Arabidopsis with the bicistronic vector carrying GFP as the fluorescent marker and, separately, the integral plasma membrane protein SYP121 essential for the inward K+ channel current. We demonstrate that transient expression of SYP121 in syp121 mutant plants is sufficient to rescue the K+ current in vivo. The combination of transient expression and use of the bicistronic vector promises significant advantages for studies of membrane transport and nutrient acquisition in roots.     

拟南芥血红蛋白3的亚细胞定位

拟南芥的血红蛋白3（AtGLB 3）属于截短的血红蛋白。与拟南芥血红蛋白1相比,拟南芥血红蛋白3具有不同的起源、不同的生化特性和结构;但其功能还不清楚。蛋白质的定位与蛋白质的功能息息相关。为深入研究该基因功能,构建了拟南芥血红蛋白3基因与绿色荧光蛋白融合的植物表达载体pUCGFP/AtGLB3。利用基因枪转化法将重组载体转入洋葱表皮细胞瞬时表达,通过检测融合蛋白在洋葱表皮细胞中的分布来确定拟南芥血红蛋白3在细胞中的定位。荧光显微镜检测结果表明,AtGLB3基因表达产物主要定位在细胞膜上。     

An efficient Agrobacterium-mediated transient transformation of Arabidopsis

Agrobacterium tumefaciens-mediated transient transformation has been a useful procedure for characterization of proteins and their functions in plants, including analysis of protein-protein interactions. Agrobacterium-mediated transient transformation of Nicotiana benthamiana by leaf infiltration has been widely used due to its ease and high efficiency. However, in Arabidopsis this procedure has been challenging. Previous studies suggested that this difficulty was caused by plant immune responses triggered by perception of Agrobacterium. Here, we report a simple and robust method for Agrobacterium-mediated transient transformation in Arabidopsis. AvrPto is an effector protein from the bacterial plant pathogen Pseudomonas syringae that suppresses plant immunity by interfering with plant immune receptors. We used transgenic Arabidopsis plants that conditionally express AvrPto under the control of a dexamethasone (DEX)-inducible promoter. When the transgenic plants were pretreated with DEX prior to infection with Agrobacterium carrying a β-glucuronidase (GUS, uidA) gene with an artificial intron and driven by the CaMV 35S promoter, transient GUS expression was dramatically enhanced compared to that in mock-pretreated plants. This transient expression system was successfully applied to analysis of the subcellular localization of a cyan fluorescent protein (CFP) fusion and a protein-protein interaction in Arabidopsis. Our findings enable efficient use of Agrobacterium-mediated transient transformation in Arabidopsis thaliana.     

High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions

We describe a streamlined and systematic method for cloning green fluorescent protein (GFP)-open reading frame (ORF) fusions and assessing their subcellular localization in Arabidopsis thaliana cells. The sequencing of the Arabidopsis genome has made it feasible to undertake genome-based approaches to determine the function of each protein and define its subcellular localization. This is an essential step towards full functional analysis. The approach described here allows the economical handling of hundreds of expressed plant proteins in a timely fashion. We have integrated recombinational cloning of full-length trimmed ORF clones (available from the SSP consortium) with high-efficiency transient transformation of Arabidopsis cell cultures by a hypervirulent strain of Agrobacterium. To demonstrate its utility, we have used a selection of trimmed ORFs, representing a variety of key cellular processes and have defined the localization patterns of 155 fusion proteins. These patterns have been classified into five main categories, including cytoplasmic, nuclear, nucleolar, organellar and endomembrane compartments. Several genes annotated in GenBank as unknown have been ascribed a protein localization pattern. We also demonstrate the application of flow cytometry to estimate the transformation efficiency and cell cycle phase of the GFP-positive cells. This approach can be extended to functional studies, including the precise cellular localization and the prediction of the role of unknown proteins, the confirmation of bioinformatic predictions and proteomic experiments, such as the determination of protein interactions in vivo, and therefore has numerous applications in the post-genomic analysis of protein function.     

Vacuolar system distribution in Arabidopsis tissues,visualized using GFP fusion proteins

Green fluorescent protein (GFP) allows the direct visualization of gene expression and the subcellular localization of fusion proteins in living cells. The localization of different GFP fusion proteins in the secretory system was studied in stably transformed Arabidopsis plants cv. Wassilewskaja. Secreted GFP (SGFP) and GFP retained in the ER (GFP-KDEL) confirmed patterns already known, but two vacuolar GFPs (GFP-Chi and Aleu-GFP) labelled the Arabidopsis vacuolar system for the first time, the organization of which appears to depend on cell differentiation. GFP stability in the vacuoles may depend on pH or degradation, but these vacuolar markers can, nevertheless, be used as a tool for physiological studies making these plants suitable for mutagenesis and gene-tagging experiments.     

Cellular and subcellular localization of flavin-monooxygenases involved in glucosinolate biosynthesis

Glucosinolates are amino acid-derived secondary metabolites with diverse biological activities dependent on chemical modifications of the side chain. Five flavin-monooxygenases FMO(GS-OX1-5) have recently been identified as aliphatic glucosinolate side chain modification enzymes in Arabidopsis thaliana that catalyse the generation of methylsulphinylalkyl glucosinolates, which can be hydrolysed to products with distinctive benefits for human health and plant defence. Though the localization of most aliphatic glucosinolate biosynthetic enzymes has been determined, little is known about where the side chain modifications take place despite their importance. Hence, the spatial expression pattern of FMO(GS-OX1-5) genes in Arabidopsis was investigated by expressing green fluorescent protein (GFP) and β-glucuronidase (GUS) fusion genes controlled by FMO(GS-OX1-5) promoters. The cellular compartmentation of FMO(GS-OX1) was also detected by transiently expressing a FMO(GS-OX1)-yellow fluorescent protein (YFP) fusion protein in tobacco leaves. The results showed that FMO(GS-OX1-5) were expressed basically in vascular tissues, especially in phloem cells, like other glucosinolate biosynthetic genes. They were also found in endodermis-like cells in flower stalk and epidermal cells in leaf, which is a location that has not been reported for other glucosinolate biosynthetic genes. It is suggested that the spatial expression pattern of FMO(GS-OX1-5) determines the access of enzymes to their substrate and therefore affects the glucosinolate profile. FMO(GS-OX1)-YFP fusion protein analysis identified FMO(GS-OX1) as a cytosolic protein. Together with the subcellular locations of the other biosynthetic enzymes, an integrated map of the multicompartmentalized aliphatic glucosinolate biosynthetic pathway is discussed.     

拟南芥新型脂转移蛋白AtDHyPRP1的亚细胞定位及其对灰霉菌的抗性

通过遗传转化技术研究了拟南芥脂转移蛋白AtDHyPRP1在细胞中的定位及其对真菌病原体的抗性。采用PCR方法从拟南芥Ws生态型克隆了AtDHyPRP1基因,构建产生pRI101-AN-AtDHyPRP1植物双元表达载体和pCAMBIA1302-AtDHyPRP1-GFP融合表达载体,经农杆菌介导的叶盘和浸花法得到烟草和拟南芥转基因植株。AtDHyPRP1基因能够明显增加烟草对灰霉菌的抗性,转AtDHyPRP1烟草叶片的被侵染部位有大量H2O2积累,激光共聚焦显微观察发现AtDHyPRP1蛋白定位于细胞表面。说明AtDHyPRP1蛋白在合成后被分泌到细胞外执行特殊的功能,与植物抗病防御机制有关。     

3D-SIM结构照明超分辨率显微镜实现蛋白质在植物亚细胞器内的定位

基因表达产物蛋白质的亚细胞定位是解析基因生物学功能的重要证据之一。近年来出现的超分辨率光学成像技术已成功应用于人类和动物细胞中,预示着显微成像技术继激光共聚焦技术后的又一重要进步。由于植物细胞的特殊性和成像技术的研发取向,超分辨率光学成像技术在植物细胞蛋白质亚细胞定位的应用尚未见报道。该研究利用Delta Vision OMX显微镜技术,克服了叶绿体基粒中叶绿素自发荧光与融合蛋白荧光不易区分的缺陷,解决了受分辨率局限无法将植物细胞中蛋白质在亚细胞器内可视化精确定位的技术难题,成功地将植物蔗糖合成酶Zm SUS-SH1定位在烟草表皮细胞叶绿体基粒周围。该研究同时建立了一套基于撕片制片法的简便OMX显微镜制片方法,并针对OMX显微成像技术在植物细胞中蛋白质亚细胞定位的应用进行了讨论。     

A Rapid,Highly Efficient and Economical Method of Agrobacterium-Mediated In planta Transient Transformation in Living Onion Epidermis

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.