Expression of an epitope-tagged virulence protein in Rickettsia parkeri using transposon insertion

Despite recent advances in our ability to genetically manipulate Rickettsia, little has been done to employ genetic tools to study the expression and localization of Rickettsia virulence proteins. Using a mariner-based Himar1 transposition system, we expressed an epitope-tagged variant of the actin polymerizing protein RickA under the control of its native promoter in Rickettsia parkeri, allowing the detection of RickA using commercially-available antibodies. Native RickA and epitope-tagged RickA exhibited similar levels of expression and were specifically localized to bacteria. To further facilitate protein expression in Rickettsia, we also developed a plasmid for Rickettsia insertion and expression (pRIE), containing a variant Himar1 transposon with enhanced flexibility for gene insertion, and used it to generate R. parkeri strains expressing diverse fluorescent proteins. Expression of epitope-tagged proteins in Rickettsia will expand our ability to assess the regulation and function of important virulence factors.     

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.     

The Arabidopsis class VIII myosin ATM2 is involved in endocytosis

Members of the class XI of the myosin superfamily comprising higher plant, actin-based molecular motors have been shown to be involved in peroxisome and Golgi vesicle trafficking comparable to yeast and animal class V myosins. The tasks of the second class of myosins of higher plants, class VIII, are unclear. In this study the class VIII myosin ATM2 from the model plant Arabidopsis thaliana was selected for the examination of cargo specificity in vivo. Fluorescent protein-fusion plasmid constructs with fragments of the ATM2 cDNA were generated and used for Agrobacterium tumefaciens-based transient transformation of Nicotiana benthamiana leaves. The resulting subcellular localization patterns were recorded by live imaging with confocal laser scanning microscopy (CLSM) in epidermal leaf cells. Expression of a nearly full-length construct displayed labeling of filaments and vesicles, a head + neck fragment led to decoration of filaments only. However, expression of fluorescent protein-tagged C-terminal tail domain constructs labeled vesicular structures of different appearance. Most importantly, coexpression of different RFP/YFP-ATM2 tail fusion proteins showed colocalization and, hence, binding to the same type of vesicular target. Further coexpression experiments of RFP/YFP-ATM2 tail fusion proteins with the endosomal marker FYVE and the endosomal tracer FM4-64 demonstrated colocalization with endosomes. Colocalization was also detected by expression of the CFP-tagged membrane receptor BRI1 as marker, which is constantly recycled via endosomes. Occasionally the ATM2 tail targeted to sites at the plasma membrane closely resembling the pattern obtained upon expression of the YFP-ATM1 C-terminal tail. ATM1 is known for its localization at the plasma membrane at sites of plasmodesmata.     

Cloning trap for signal peptide sequences

Novel secreted and/or type I transmembrane proteins containing N-terminal signal sequences have been successfully cloned using the signal sequence trapping (SST) method. Often this involves random cloning of short 5' cDNA terminal ends into an epitope-tagged expression vector and the detection of expressed recombinant proteins on the cell surfaces of transfected cells with an antibody to the tagged epitope. Here, we report a novel cloning system for the detection of secreted proteins also using SST. In this method, we used the human immunodeficiency virus (HIV-1) p24 as the epitope for tagging. To test the system, two constructs were created. The 5' terminal end of a human beta-chemokine (which was regulated upon activation, expressed by normal T cells and presumably secreted [RANTES]) and the 5' end of a human CD4 receptor were cloned upstream of and in-frame with the p24 cDNA. Secreted p24 was detectable in the culture media two days after transfection of either DNA construct into the human cell lines, HeLa and 293T. When the chimeric p24 expression constructs were transfected at a ratio of 1:100 to the vector pcDNA3.1(+), p24 could still be detected in cell supernatants. The use of a secreted viral antigen like HIV-1 p24 (or of any noncellular protein) as a marker in SST cloning approaches is likely to be advantageous because it reduces the background noise in detection and also renders this system suitable for high-throughput screening.     

Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defense gene expression

Agrobacterium tumefaciens is a plant pathogen that incites crown gall tumors by transferring to and expressing a portion of a resident plasmid in plant cells. Currently, little is known about the host response to Agrobacterium infection. Using suppressive subtractive hybridization and DNA macroarrays, we identified numerous plant genes that are differentially expressed during early stages of Agrobacterium-mediated transformation. Expression profiling indicates that Agrobacterium infection induces plant genes necessary for the transformation process while simultaneously repressing host defense response genes, thus indicating successful utilization of existing host cellular machinery for genetic transformation purposes. A comparison of plant responses to different strains of Agrobacterium indicates that transfer of both T-DNA and Vir proteins modulates the expression of host genes during the transformation process.     

Expression of the Arabidopsis histone H2A-1 gene correlates with susceptibility to Agrobacterium transformation

Transformation of plant cells by Agrobacterium tumefaciens involves both bacterial virulence proteins and host proteins. We have previously shown that the Arabidopsis thaliana gene H2A-1 (RAT5), which encodes histone H2A-1, is involved in T-DNA integration into the plant genome. Mutation of RAT5 results in a severely decreased frequency of transformation, whereas overexpression of RAT5 enhances the transformation frequency (Mysore et al., 2000b). We show here that the expression pattern of RAT5 correlates with plant root cells most susceptible to transformation. As opposed to a cyclin-GUS fusion gene whose expression is limited to meristematic tissues, the H2A-1 gene is expressed in many non-dividing cells. Under normal circumstances, the H2A-1 gene is expressed in the elongation zone of the root, the region that is most susceptible to Agrobacterium transformation. In addition, when roots are incubated on medium containing phytohormones, a concomitant shift in H2A-1 expression and transformation susceptibility to the root base is observed. Inoculation of root segments with a transfer-competent, but not a transformation-deficient Agrobacterium strain induces H2A-1 expression. Furthermore, pre-treatment of Arabidopsis root segments with phytohormones both induces H2A-1 expression and increases the frequency of Agrobacterium transformation. Our results suggest that the expression of the H2A-1 gene is both a marker for, and a predictor of, plant cells most susceptible to Agrobacterium transformation.     

Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.     

VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity.

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. This event is thought to be mediated by two bacterial proteins, VirD2 and VirE2, which are associated with the transported T-DNA molecule. While VirD2 is imported into the nuclei of plant, animal and yeast cells, nuclear uptake of VirE2 occurs most efficiently in plant cells. To understand better the mechanism of VirE2 action, a cellular interactor of VirE2 was identified and its encoding gene cloned from Arabidopsis. The identified plant protein, designated VIP1, specifically bound VirE2 and allowed its nuclear import in non-plant systems. In plants, VIP1 was required for VirE2 nuclear import and Agrobacterium tumorigenicity, participating in early stages of T-DNA expression.     

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDV-m) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three- to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals.     

Characterization of Agrobacterium tumefaciens virulence proteins induced by the plant factor acetosyringone

The Ti plasmid virulence (vir) loci encode functions essential for the transfer of the T-DNA element from Agrobacterium tumefaciens to plant cells. The expression of these loci is specifically signaled by plant phenolics such as acetosyringone. Here, we characterize the protein products that are induced in Agrobacterium grown in the presence of acetosyringone. More than 10 to 15 proteins are induced in strains harboring different Ti plasmids. Two general classes of acetosyringone-induced proteins are observed, encoded either within or outside the vir region. Synthesis of both classes of proteins requires acetosyringone and the products of the vir regulatory genes A and G. Those proteins encoded outside the vir region define a novel category of proteins, the virulence-related proteins, which are both chromosomally and Ti plasmid-encoded. The molecular weight and subcellular localization of several pTiA6 vir-induced proteins are identified. The most abundant induced protein has a molecular weight of 65,000, and is the single product of the virE locus; this protein distributes into both cell envelope and soluble fractions. Three proteins with molecular weights of approximately 33,000, 80,000 and 25,000 fractionate with the cell envelope and are encoded by genes within the 5' half of the virB locus. The envelope localization of the virB proteins suggests that they play a role in directing T-DNA transfer events that occur at the bacterial surface.     

Interaction between <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> oncoprotein 6b and a tobacco nucleolar protein that is homologous to TNP1 encoded by a transposable element of <Emphasis Type="Italic">Antirrhinum majus</Emphasis>

When gene 6b on the T-DNA of Agrobacterium tumefaciens is transferred to plant cells, its expression causes plant hormone-independent division of cells in in vitro culture and abnormal cell growth, which induces various morphological defects in 6b-expressing transgenic Arabidopsis thaliana and Nicotiana tabacum plants. Protein 6b localizes to the nuclei, a requirement for the abnormal cell growth, and binds to a tobacco nuclear protein called NtSIP1 and histone H3. In addition, 6b has histone chaperone-like activity in vitro and affects the expression of various plant genes, including cell division-related genes and meristem-related class 1 KNOX homeobox genes, in transgenic Arabidopsis. Here, we report that 6b binds to a newly identified protein NtSIP2, whose amino acid sequence is predicted to be 30% identical and 51% similar to that of the TNP1 protein encoded by the transposon Tam1 of Antirrhinum majus. Immunolocalization analysis using anti-T7 antibodies showed nucleolar localization of most of the T7 epitope-tagged NtSIP2 proteins. A similar analysis with the T7-tagged 6b protein also showed subnucleolar as well as nuclear localization of the 6b protein. These results suggest the involvement of 6b along with NtSIP2 in certain molecular processes in the nucleolus as well as the nucleoplasm.     

Agrobacterium rhizogenes GALLS protein contains domains for ATP binding, nuclear localization, and type IV secretion

Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2.     

Plant proteins that interact with VirB2, the Agrobacterium tumefaciens pilin protein, mediate plant transformation

Agrobacterium tumefaciens uses a type IV secretion system (T4SS) to transfer T-DNA and virulence proteins to plants. The T4SS is composed of two major structural components: the T-pilus and a membrane-associated complex that is responsible for translocating substrates across both bacterial membranes. VirB2 protein is the major component of the T-pilus. We used the C-terminal-processed portion of VirB2 protein as a bait to screen an Arabidopsis thaliana cDNA library for proteins that interact with VirB2 in yeast. We identified three related plant proteins, VirB2-interacting protein (BTI) 1 (BTI1), BTI2, and BTI3 with unknown functions, and a membrane-associated GTPase, AtRAB8. The three BTI proteins also interacted with VirB2 in vitro. Preincubation of Agrobacterium with GST-BTI1 protein decreased the transformation efficiency of Arabidopsis suspension cells by Agrobacterium. Transgenic BTI and AtRAB8 antisense and RNA interference Arabidopsis plants are less susceptible to transformation by Agrobacterium than are wild-type plants. The level of BTI1 protein is transiently increased immediately after Agrobacterium infection. In addition, overexpression of BTI1 protein in transgenic Arabidopsis results in plants that are hypersusceptible to Agrobacterium-mediated transformation. Confocal microscopic data indicate that GFP-BTI proteins preferentially localize to the periphery of root cells in transgenic Arabidopsis plants, suggesting that BTI proteins may contact the Agrobacterium T-pilus. We propose that the three BTI proteins and AtRAB8 are involved in the initial interaction of Agrobacterium with plant cells.     

The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation

To genetically transform plants, Agrobacterium exports its transferred DNA (T-DNA) and several virulence (Vir) proteins into the host cell. Among these proteins, VirE3 is the only one whose biological function is completely unknown. Here, we demonstrate that VirE3 is transferred from Agrobacterium to the plant cell and then imported into its nucleus via the karyopherin alpha-dependent pathway. In addition to binding plant karyopherin alpha, VirE3 interacts with VirE2, a major bacterial protein that directly associates with the T-DNA and facilitates its nuclear import. The VirE2 nuclear import in turn is mediated by a plant protein, VIP1. Our data indicate that VirE3 can mimic this VIP1 function, acting as an 'adapter' molecule between VirE2 and karyopherin alpha and 'piggy-backing' VirE2 into the host cell nucleus. As VIP1 is not an abundant protein, representing one of the limiting factors for transformation, Agrobacterium may have evolved to produce and export to the host cells its own virulence protein that at least partially complements the cellular VIP1 function necessary for the T-DNA nuclear import and subsequent expression within the infected cell.     

Protein mislocalization in plant cells using a GFP‐binding chromobody

A key challenge in cell biology is to directly link protein localization to function. The green fluorescent protein (GFP)‐binding protein, GBP, is a 13‐kDa soluble protein derived from a llama heavy chain antibody that binds with high affinity to GFP as well as to some GFP variants such as yellow fluorescent protein (YFP). A GBP fusion to the red fluorescent protein (RFP), a molecule termed a chromobody, was previously used to trace in vivo the localization of various animal antigens. In this study, we extend the use of chromobody technology to plant cells and develop several applications for the in vivo study of GFP‐tagged plant proteins. We took advantage of Agrobacterium tumefaciens‐mediated transient expression assays (agroinfiltration) and virus expression vectors (agroinfection) to express functional GBP:RFP fusion (chromobody) in the model plant Nicotiana benthamiana. We showed that the chromobody is effective in binding GFP‐ and YFP‐tagged proteins in planta. Most interestingly, GBP:RFP can be applied to interfere with the function of GFP fusion protein and to mislocalize (trap) GFP fusions to the plant cytoplasm in order to alter the phenotype mediated by the targeted proteins. Chromobody technology, therefore, represents a new alternative technique for protein interference that can directly link localization of plant proteins to in vivo function.     

Root phloem-specific expression of the plasma membrane amino acid proton co-transporter AAP3

Amino acids are regarded as the nitrogen 'currency' of plants. Amino acids can be taken up from the soil directly or synthesized from inorganic nitrogen, and then circulated in the plant via phloem and xylem. AtAAP3, a member of the Amino Acid Permease (AAP) family, is mainly expressed in root tissue, suggesting a potential role in the uptake and distribution of amino acids. To determine the spatial expression pattern of AAP3, promoter-reporter gene fusions were introduced into Arabidopsis. Histochemical analysis of AAP3 promoter-GUS expressing plants revealed that AAP3 is preferentially expressed in root phloem. Expression was also detected in stamens, in cotyledons, and in major veins of some mature leaves. GFP-AAP3 fusions and epitope-tagged AAP3 were used to confirm the tissue specificity and to determine the subcellular localization of AtAAP3. When overexpressed in yeast or plant protoplasts, the functional GFP-AAP3 fusion was localized in subcellular organelle-like structures, nuclear membrane, and plasma membrane. Epitope-tagged AAP3 confirmed its localization to the plasma membrane and nuclear membrane of the phloem, consistent with the promoter-GUS study. In addition, epitope-tagged AAP3 protein was localized in endodermal cells in root tips. The intracellular localization suggests trafficking or cycling of the transporter, similar to many metabolite transporters in yeast or mammals, for example, yeast amino acid permease GAP1. Despite the specific expression pattern, knock-out mutants did not show altered phenotypes under various conditions including N-starvation. Microarray analyses revealed that the expression profile of genes involved in amino acid metabolism did not change drastically, indicating potential compensation by other amino acid transporters.     

The Arabidopsis rab5 homologs rha1 and ara7 localize to the prevacuolar compartment

Rha1, an Arabidopsis Rab5 homolog, plays a critical role in vacuolar trafficking in plant cells. In this study, we investigated the localization of Rha1 and Ara7, two Arabidopsis proteins that have highly similar amino acid sequence homology to Rab5 in animal cells. Both Ara7 and Rha1 gave a punctate staining pattern and colocalized when transiently expressed as GFP- (green fluorescent protein) or small epitope-tagged forms in Arabidopsis protoplasts. In protoplasts, transiently expressed Rha1 and Ara7 colocalized with AtPEP12p and VSR(At-1), two proteins that are known to be present at the prevacuolar compartment (PVC). Furthermore, endogenous Rha1 also gave a punctate staining pattern and colocalized with AtPEP12p to the PVC. Mutations in the first and second GTP-binding motifs alter the localizations of GFP: Rha1[S24N] in the cytosol and Rha1[Q69L] in the tonoplast of the central vacuole. Also, mutations in the effector domain and the prenylation site inhibit membrane association of Rha1. Based on these results, we propose that Rha1 and Ara7 localize to the PVC and that GTP-binding motifs as well as the effector domain are important for localization of Rha1 to the PVC.