Restriction-ligation-independent production of a TVCV infectious clone and a TVCV-based gene expression vector.

Transgenic expression of proteins in plants is central to research and biotechnology, and, often, it is desirable to obtain this expression without altering the nuclear or plastid genomes. Thus, expression vectors based on plant viruses that infect multiple cells are useful; furthermore, they are also advantageous for studies of the life cycle of the virus itself. Here, we report the development of an expression vector based on a Turnip vein-clearing virus (TVCV), a tobamovirus known to easily infect two model plants, Nicotiana benthamiana, and Arabidopsis thaliana. Avoiding restriction digestion, we utilized a restriction-ligation-independent cloning approach to construct an infectious cDNA clone of TVCV from the viral RNA and then to convert this clone to a gene expression vector adapted for Gateway-based recombination cloning for transgene insertion. The functionality of the resulting vector, designated pTVCV-DEST, was validated by the expression of an autofluorescent reporter transgene following agroinoculation of the target plant.

Arabidopsis LSH10 transcription factor and OTLD1 histone deubiquitinase interact and transcriptionally regulate the same target genes.

Histone ubiquitylation/deubiquitylation plays a major role in the epigenetic regulation of gene expression. In plants, OTLD1, a member of the ovarian tumor (OTU) deubiquitinase family, deubiquitylates histone 2B and represses the expression of genes involved in growth, cell expansion, and hormone signaling. OTLD1 lacks the intrinsic ability to bind DNA. How OTLD1, as well as most other known plant histone deubiquitinases, recognizes its target genes remains unknown. Here, we show that Arabidopsis transcription factor LSH10, a member of the ALOG protein family, interacts with OTLD1 in living plant cells. Loss-of-function LSH10 mutations relieve the OTLD1-promoted transcriptional repression of the target genes, resulting in their elevated expression, whereas recovery of the LSH10 function results in down-regulated transcription of the same genes. We show that LSH10 associates with the target gene chromatin as well as with DNA sequences in the promoter regions of the target genes. Furthermore, without LSH10, the degree of H2B monoubiquitylation in the target promoter chromatin increases. Hence, our data suggest that OTLD1-LSH10 acts as a co-repressor complex potentially representing a general mechanism for the specific function of plant histone deubiquitinases at their target chromatin.

Gain-of-function mutant of movement protein allows systemic transport of a defective tobacco mosaic virus.

Functional compensation in response to gene dysfunction is a fascinating phenomenon that allows mutated viruses to regain the capabilities of their wild-type parental strains. In this study, we isolated mutants of tobacco mosaic virus capable of CP-independent systemic movement. These gain-of-function mutants lacked the 16 C-terminal amino acids of the movement protein (MP). Whereas this deletion did not affect the cell-to-cell movement of MP, it dramatically enhanced the viral genomic RNA levels and MP accumulation within the infected cells and altered the subcellular localization of MP from exclusively plasmodesmata (PD) to both PD and plasma membrane. The adapted defective virus suppressed the expression of the ethylene pathway and phloem-associated resistance factors in the inoculated leaves. These findings demonstrate the potential for plant viral MPs to gain a new function that allows viral genomes to move systemically in the absence of the natural viral factor that mediates this spread.

Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer.

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone marks for transcriptional repression or activation, respectively. HMEs are recruited to their target chromatin by transcription factors (TFs). Thus, detecting and characterizing direct interactions between HMEs and TFs are critical for understanding their function and specificity better. These studies would be more biologically relevant if performed in vivo within living tissues. Here, a protocol is described for visualizing interactions in plant leaves between a plant histone deubiquitinase and a plant transcription factor using fluorescence resonance energy transfer (FRET), which allows the detection of complexes between protein molecules that are within <10 nm from each other. Two variations of the FRET technique are presented: SE-FRET (sensitized emission) and AB-FRET (acceptor bleaching), in which the energy is transferred non-radiatively from the donor to the acceptor or emitted radiatively by the donor upon photobleaching of the acceptor. Both SE-FRET and AB-FRET approaches can be adapted easily to discover other interactions between other proteins in planta.

Genetic factors governing bacterial virulence and host plant susceptibility during Agrobacterium infection.

Several species of the Agrobacterium genus represent unique bacterial pathogens able to genetically transform plants, by transferring and integrating a segment of their own DNA (T-DNA, transferred DNA) in their host genome. Whereas in nature this process results in uncontrolled growth of the infected plant cells (tumors), this capability of Agrobacterium has been widely used as a crucial tool to generate transgenic plants, for research and biotechnology. The virulence of Agrobacterium relies on a series of virulence genes, mostly encoded on a large plasmid (Ti-plasmid, tumor inducing plasmid), involved in the different steps of the DNA transfer to the host cell genome: activation of bacterial virulence, synthesis and export of the T-DNA and its associated proteins, intracellular trafficking of the T-DNA and effector proteins in the host cell, and integration of the T-DNA in the host genomic DNA. Multiple interactions between these bacterial encoded proteins and host factors occur during the infection process, which determine the outcome of the infection. Here, we review our current knowledge of the mechanisms by which bacterial and plant factors control Agrobacterium virulence and host plant susceptibility.

Receptor-like kinase BAM1 facilitates early movement of the Tobacco mosaic virus.

Cell-to-cell movement is an important step for initiation and spreading of virus infection in plants. This process occurs through the intercellular connections, termed plasmodesmata (PD), and is usually mediated by one or more virus-encoded movement proteins (MP) which interact with multiple cellular factors, among them protein kinases that usually have negative effects on MP function and virus movement. In this study, we report physical and functional interaction between MP of Tobacco mosaic virus (TMV), the paradigm of PD-moving proteins, and a receptor-like kinase BAM1 from Arabidopsis and its homolog from Nicotiana benthamiana. The interacting proteins accumulated in the PD regions, colocalizing with a PD marker. Reversed genetics experiments, using BAM1 gain-of-function and loss-of-function plants, indicated that BAM1 is required for efficient spread and accumulation the virus during initial stages of infection of both plant species by TMV. Furthermore, BAM1 was also required for the efficient cell-to-cell movement of TMV MP, suggesting that BAM1 interacts with TMV MP to support early movement of the virus. Interestingly, this role of BAM1 in viral movement did not require its protein kinase activity. Thus, we propose that association of BAM1 with TMV MP at PD facilitates the MP transport through PD, which, in turn, enhances the spread of the viral infection.

Modulation of plant DNA damage response gene expression during Agrobacterium infection.

Agrobacterium T-DNA (transfer DNA) integration into the plant genome relies mostly on host proteins involved in the DNA damage repair pathways. However, conflicting results have been obtained using plants with mutated or down-regulated genes involved in these pathways. Here, we chose a different approach by following the expression of a series of genes, encoding proteins involved in the DNA damage response, during early stages of Agrobacterium infection in tobacco. First, we identified tobacco homologs of Arabidopsis genes induced upon DNA damage and demonstrated that their expression was activated by bleomycin, a DNA-break causing agent. Then, we showed that Agrobacterium infection induces the expression of several of these genes markers of the host DNA damage response, with different patterns of transcriptional response. This induction largely depends on Agrobacterium virulence factors, but not on the T-DNA, suggesting that the DNA damage response activation may rely on Agrobacterium-encoded virulence proteins. Our results suggest that Agrobacterium modulates the plant DNA damage response machinery, which might facilitate the integration of the bacterial T-DNA into the DNA breaks in the host genome.

Rapid generation of inoculum of a plant RNA virus using overlap PCR.

We have developed an efficient method to rapidly generate infectious inoculum of a plant RNA virus and confirmed its infectivity by mechanical inoculation. The method takes advantage of overlap PCR to bypass the cloning steps, which makes it relatively simple, rapid, and inexpensive compared to the traditional methods. Using this approach, inoculum of a tobamovirus, Turnip vein clearing virus (TVCV), was generated. PCR products specific for the 35S promoter and TVCV genome were used as templates for overlap PCR to form a single product containing the full-length TVCV cDNA under the control of the double 35S promoter, and the entire process took only 8 h. This inoculum was infectious in Nicotiana benthamiana, and its infectivity was ca. 67% compared to 0% and 100% with negative and positive controls, respectively. Thus, this rapid method generates efficient infectious inoculum for a plant RNA virus.

Biolistic Approach for Transient Gene Expression Studies in Plants.

Since its inception in the late 1980s, the delivery of exogenous nucleic acids into living cells via high-velocity microprojectiles (biolistic, or microparticle bombardment) has been an invaluable tool for both agricultural and fundamental plant research. Here, we review the technical aspects and the major applications of the biolistic method for studies involving transient gene expression in plant cells. These studies cover multiple areas of plant research, including gene expression, protein subcellular localization and cell-to-cell movement, plant virology, silencing, and the more recently developed targeted genome editing via transient expression of customized endonucleases.

Histone Deubiquitinase OTU1 Epigenetically Regulates DA1 and DA2, Which Control Arabidopsis Seed and Organ Size.

Seeds are central to plant life cycle and to human nutrition, functioning as the major supplier of human population energy intake. To understand better the roles of enzymic writers and erasers of the epigenetic marks, in particular, histone ubiquitylation and the corresponding histone modifiers, involved in control of seed development, we identified the otubain-like cysteine protease OTU1 as a histone deubiquitinase involved in transcriptional repression of the DA1 and DA2 genes known to regulate seed and organ size in Arabidopsis. Loss-of-function mutants of OTU1 accumulate H2B monoubiquitylation and such euchromatic marks as H3 trimethylation and hyperacetylation in the DA1 and DA2 chromatin. These data advance our knowledge about epigenetic regulation of the DA1 and DA2 genes by recognizing OTU1 as a member of a putative repressor complex that negatively regulates their transcription.

Pathways of DNA Transfer to Plants from Agrobacterium tumefaciens and Related Bacterial Species.

Genetic transformation of host plants by Agrobacterium tumefaciens and related species represents a unique model for natural horizontal gene transfer. Almost five decades of studying the molecular interactions between Agrobacterium and its host cells have yielded countless fundamental insights into bacterial and plant biology, even though several steps of the DNA transfer process remain poorly understood. Agrobacterium spp. may utilize different pathways for transferring DNA, which likely reflects the very wide host range of Agrobacterium. Furthermore, closely related bacterial species, such as rhizobia, are able to transfer DNA to host plant cells when they are provided with Agrobacterium DNA transfer machinery and T-DNA. Homologs of Agrobacterium virulence genes are found in many bacterial genomes, but only one non-Agrobacterium bacterial strain, Rhizobium etli CFN42, harbors a complete set of virulence genes and can mediate plant genetic transformation when carrying a T-DNA-containing plasmid.

Coordinate activation of a target gene by KDM1C histone demethylase and OTLD1 histone deubiquitinase in Arabidopsis.

Potential functional coordination between histone deubiquitinases and histone lysine demethylases represents one of the least studied aspects of epigenetic control of transcriptional outcomes. Here, this question was addressed using Arabidopsis histone modification erasers deubiquitinase OTLD1 and demethylase KDM1C known to interact with each other in plant cells. Characterization of gain- and loss-of-function mutants of OTLD1 and KDM1C showed that both enzymes associate with the promoter chromatin of their target gene AN3 and function as coactivators of its expression. This transcriptional outcome was underlain by demethylation of the H3K9 repression mark, presumably by the KDM1C histone demethylase activity. Association of KDM1C and OTLD1 with the target chromatin was interdependent such that OTLD1 was not detected at the AN3 in the absence of KDM1C and KDM1C displayed a different and non-functional pattern of association in the absence of OTLD1. Thus, OTLD1 and KDM1C may crosstalk with each other to assemble a functional coactivator complex at the AN3 promoter chromatin and set the KDM1C specificity for the methylated H3K9 to determine the correct transcriptional outcome.

The Plasmodesmal Localization Signal of TMV MP Is Recognized by Plant Synaptotagmin SYTA.

Plant viruses cross the barrier of the plant cell wall by moving through intercellular channels, termed plasmodesmata, to invade their hosts. They accomplish this by encoding movement proteins (MPs), which act to alter plasmodesmal gating. How MPs target to plasmodesmata is not well understood. Our recent characterization of the first plasmodesmal localization signal (PLS) identified in a viral MP, namely, the MP encoded by the Tobamovirus Tobacco mosaic virus (TMV), now provides the opportunity to identify host proteins that recognize this PLS and may be important for its plasmodesmal targeting. One such candidate protein is Arabidopsis synaptotagmin A (SYTA), which is required to form endoplasmic reticulum (ER)-plasma membrane contact sites and regulates the MP-mediated trafficking of begomoviruses, tobamoviruses, and potyviruses. In particular, SYTA interacts with, and regulates the cell-to-cell transport of, both TMV MP and the MP encoded by the Tobamovirus Turnip vein clearing virus (TVCV). Using in planta bimolecular fluorescence complementation (BiFC) and yeast two-hybrid assays, we show here that the TMV PLS interacted with SYTA. This PLS sequence was both necessary and sufficient for interaction with SYTA, and the plasmodesmal targeting activity of the TMV PLS was substantially reduced in an Arabidopsis syta knockdown line. Our findings show that SYTA is one host factor that can recognize the TMV PLS and suggest that this interaction may stabilize the association of TMV MP with plasmodesmata.IMPORTANCE Plant viruses use their movement proteins (MPs) to move through host intercellular connections, plasmodesmata. Perhaps one of the most intriguing, yet least studied, aspects of this transport is the MP signal sequences and their host recognition factors. Recently, we have described the plasmodesmal localization signal (PLS) of the Tobacco mosaic virus (TMV) MP. Here, we identified the Arabidopsis synaptotagmin A (SYTA) as a host factor that recognizes TMV MP PLS and promotes its association with the plasmodesmal membrane. The significance of these findings is two-fold: (i) we identified the TMV MP association with the cell membrane at plasmodesmata as an important PLS-dependent step in plasmodesmal targeting, and (ii) we identified the plant SYTA protein that specifically recognizes PLS as a host factor involved in this step.

Beyond Agrobacterium-Mediated Transformation: Horizontal Gene Transfer from Bacteria to Eukaryotes.

Besides the massive gene transfer from organelles to the nuclear genomes, which occurred during the early evolution of eukaryote lineages, the importance of horizontal gene transfer (HGT) in eukaryotes remains controversial. Yet, increasing amounts of genomic data reveal many cases of bacterium-to-eukaryote HGT that likely represent a significant force in adaptive evolution of eukaryotic species. However, DNA transfer involved in genetic transformation of plants by Agrobacterium species has traditionally been considered as the unique example of natural DNA transfer and integration into eukaryotic genomes. Recent discoveries indicate that the repertoire of donor bacterial species and of recipient eukaryotic hosts potentially are much wider than previously thought, including donor bacterial species, such as plant symbiotic nitrogen-fixing bacteria (e.g., Rhizobium etli) and animal bacterial pathogens (e.g., Bartonella henselae, Helicobacter pylori), and recipient species from virtually all eukaryotic clades. Here, we review the molecular pathways and potential mechanisms of these trans-kingdom HGT events and discuss their utilization in biotechnology and research.

The Agrobacterium VirE2 effector interacts with multiple members of the Arabidopsis VIP1 protein family.

T-DNA transfer from Agrobacterium to its host plant genome relies on multiple interactions between plant proteins and bacterial effectors. One such plant protein is the Arabidopsis VirE2 interacting protein (AtVIP1), a transcription factor that binds Agrobacterium tumefaciens C58 VirE2, potentially acting as an adaptor between VirE2 and several other host factors. It remains unknown, however, whether the same VirE2 protein has evolved to interact with multiple VIP1 homologues in the same host, and whether VirE2 homologues encoded by different bacterial strains/species recognize AtVIP1 or its homologues. Here, we addressed these questions by systematic analysis (using the yeast two-hybrid and co-immunoprecipitation approaches) of interactions between VirE2 proteins encoded by four major representatives of known bacterial species/strains with functional T-DNA transfer machineries and eight VIP1 homologues from Arabidopsis and tobacco. We also analysed the determinants of the VirE2 sequence involved in these interactions. These experiments showed that the VirE2 interaction is degenerate: the same VirE2 protein has evolved to interact with multiple VIP1 homologues in the same host, and different and mutually independent VirE2 domains are involved in interactions with different VIP1 homologues. Furthermore, the VIP1 functionality related to the interaction with VirE2 is independent of its function as a transcriptional regulator. These observations suggest that the ability of VirE2 to interact with VIP1 homologues is deeply ingrained into the process of Agrobacterium infection. Indeed, mutations that abolished VirE2 interaction with AtVIP1 produced no statistically significant effects on interactions with VIP1 homologues or on the efficiency of genetic transformation.

The Agrobacterium F-Box Protein Effector VirF Destabilizes the Arabidopsis GLABROUS1 Enhancer/Binding Protein-Like Transcription Factor VFP4, a Transcriptional Activator of Defense Response Genes.

Agrobacterium-mediated genetic transformation not only represents a technology of choice to genetically manipulate plants, but it also serves as a model system to study mechanisms employed by invading pathogens to counter the myriad defenses mounted against them by the host cell. Here, we uncover a new layer of plant defenses that is targeted by A. tumefaciens to facilitate infection. We show that the Agrobacterium F-box effector VirF, which is exported into the host cell, recognizes an Arabidopsis transcription factor VFP4 and targets it for proteasomal degradation. We hypothesize that VFP4 resists Agrobacterium infection and that the bacterium utilizes its VirF effector to degrade VFP4 and thereby mitigate the VFP4-based defense. Indeed, loss-of-function mutations in VFP4 resulted in differential expression of numerous biotic stress-response genes, suggesting that one of the functions of VFP4 is to control a spectrum of plant defenses, including those against Agrobacterium tumefaciens. We identified one such gene, ATL31, known to mediate resistance to bacterial pathogens. ATL31 was transcriptionally repressed in VFP4 loss-of-function plants and activated in VFP4 gain-of-function plants. Gain-of-function lines of VFP4 and ATL31 exhibited recalcitrance to Agrobacterium tumorigenicity, suggesting that A. tumefaciens may utilize the host ubiquitin/proteasome system to destabilize transcriptional regulators of the host disease response machinery.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta.

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Activation of gene expression by histone deubiquitinase OTLD1.

One of the main mechanisms of epigenetic control is post translational modification of histones, and one of the relatively less characterized, yet functionally important histone modifications is monoubiquitylation, which is reversed by histone deubiquitinases. In Arabidopsis, only two of such enzymes are known to date. One of them, OTLD1, deubiquitylates histone 2B and functions as a transcriptional repressor. But, could the same deubiquitinase act both as a repressor and an activator? Here, we addressed this question. Using gain-of-function and loss-of-function Arabidopsis alleles, we showed that OTLD1 can promote expression of a target gene. This transcriptional activation activity of OTLD1 involves occupation of the target chromatin by this enzyme, deubiquitination of monoubiquitylated H2B within the occupied regions, and formation of the euchromatic histone acetylation and methylation marks. Thus, OTLD1 can play a dual role in transcriptional repression and activation of its target genes. In these reactions, H2B ubiquitylation acts as both a repressive and an active mark whereas OTLD1 association with and deubiquitylation of the target chromatin may represent the key juncture between two opposing effects of this enzyme on gene expression.