Systemic transport of RNA in plants.
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Biological Transport↗
Biomedical subjects
P Zambryski
Publications and source records attributed to P Zambryski.
The transfer of DNA from agrobacterium tumefaciens into plants: a feast of fundamental insights.
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Agrobacterium tumefaciens↗
The N- and C-terminal portions of the Agrobacterium VirB1 protein independently enhance tumorigenesis.
Genetic transformation of plants by Agrobacterium tumefaciens is mediated by a virulence (vir)-specific type IV secretion apparatus assembled from 11 VirB proteins and VirD4. VirB1, targeted to the periplasm by an N-terminal signal peptide, is processed to yield VirB1*, comprising the C-terminal 73 amino acids. The N-terminal segment, which shares homology with chicken egg white lysozyme as well as lytic transglycosylases, may provide local lysis of the peptidoglycan cell wall to create channels for transporter assembly. Synthesis of VirB1* followed by its secretion to the exterior of the cell suggests that VirB1* may also have a role in virulence. In the present study, we provide evidence for the dual roles of VirB1 in tumorigenesis as well as the requirements for processing and secretion of VirB1*. Complementation of a virB1 deletion strain with constructs expressing either the N-terminal lysozyme-homologous region or VirB1* results in tumors intermediate in size between those induced by a wild-type strain and a virB1 deletion strain, suggesting that each domain has a unique role in tumorigenesis. The secretion of VirB1* translationally fused to the signal peptide indicates that processing and secretion are not coupled. When expressed independently of all other vir genes, VirB1 was processed and VirB1* was secreted. When restricted to the cytoplasm by deletion of the signal peptide, VirB1 was neither processed nor secreted and did not restore virulence to the virB1 deletion strain. Thus, factors that mediate processing of VirB1 and secretion of VirB1* are localized in the periplasm or outer membrane and are not subject to vir regulation.
Agrobacterium tumefaciens↗
Plasmodesmata: gatekeepers for cell-to-cell transport of developmental signals in plants.
Cell walls separate individual plant cells. To enable essential intercellular communication, plants have evolved membrane-lined channels, termed plasmodesmata, that interconnect the cytoplasm between neighboring cells. Historically, plasmodesmata were viewed as facilitating traffic of low-molecular weight growth regulators and nutrients critical to growth. Evidence for macromolecular transport via plasmodesmata was solely based on the exploitation of plasmodesmata by plant viruses during infectious spread. Now plasmodesmata are revealed to transport endogenous proteins, including transcription factors important for development. Two general types of proteins, non-targeted and plasmodesmata-targeted, traffic plasmodesmata channels. Size and subcellular location influence non-targeted protein transportability. Superimposed on cargo-specific parameters, plasmodesmata themselves fluctuate in aperture between closed, open, and dilated. Furthermore, plasmodesmata alter their transport capacity temporally during development and spatially in different regions of the plant. Plasmodesmata are exposed as major gatekeepers of signaling molecules that facilitate or regulate developmental programs, maintain physiological status, and respond to pathogens.
Biological Transport↗
On the origin and function of pili.
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Bacterial Adhesion↗
Assembly of the VirB transport complex for DNA transfer from Agrobacterium tumefaciens to plant cells.
The VirB transporter is a type IV secretion system that mediates the genetic transformation of plant cells by Agrobacterium tumefaciens. Assembly of this transporter depends on, first, formation of a VirB7/B9 complex that stabilizes many of the VirB proteins, second, formation of a virulence-specific pilus composed primarily of VirB2 and VirB5, and, third, post-translational processing of VirB1 and VirB2.
Agrobacterium tumefaciens↗
Plasmodesmata: gateways for information transfer.
Intercellular communication in plants has evolved to occur via elongated cytoplasmic bridges, called plasmodesmata, that traverse the thick cell walls that surround plant cells. Historically, plasmodesmata have been assigned the mostly passive role of creating cytoplasmic continuity between plant cells enabling free transport of the wealth of small plant metabolites and growth hormones under 1 kDa. When it was discovered that plant viruses pirate plasmodesmata for movement of viral genomes during infection, it was proposed that viruses modified plasmodesmata for transport of very large molecules. Now, there is compelling evidence that plasmodesmata are inherently dynamic, rapidly altering their dimensions to increase their transport capabilities, upon contact with viral as well as developmentally important plant proteins. Further, the study of intercellular transport has prompted analyses of intracellular transport pathways, implicating the cytoskeleton as a major tracking system to plasmodesmata. Thus, plasmodesmata form a three-dimensional network of transportation channels and major checkpoints for information transfer. In the following, current knowledge about structure and function of these connective organelles, and about routing of molecules towards plasmodesmata, will be summarized.
Biological Transport↗
Signaling in plant-microbe interactions.
Analysis of viral and bacterial pathogenesis has revealed common themes in the ways in which plants and animals respond to pathogenic agents. Pathogenic bacteria use macromolecule delivery systems (types III and IV) to deliver microbial avirulence proteins and transfer DNA-protein complexes directly into plant cells. The molecular events that constitute critical steps of plant-pathogen interactions seem to involve ligand-receptor mechanisms for pathogen recognition and the induction of signal transduction pathways in the plant that lead to defense responses. Unraveling the molecular basis of disease resistance pathways has laid a foundation for the rational design of crop protection strategies.
Animals↗
Ultrastructural analysis of leaf trichome plasmodesmata reveals major differences from mesophyll plasmodesmata.
Functional studies on molecular transport through plasmodesmata in leaf mesophyll and trichome cells revealed significant differences in their basal size-exclusion limits and their response to microinjected tobacco mosaic virus movement protein (E. Waigmann et al., 1994, Proc. Natl. Acad. Sci. USA 91: 1433-1437; E. Waigmann and P. Zambryski, 1995, Plant Cell 7; 2069-2079). To address the basis for these functional differences, Nicotiana clevelandii trichome and mesophyll plasmodesmata were compared ultrastructurally. Trichome plasmodesmata increase in ultrastructural complexity from the tip to the base cell. Their neck regions, thought to control molecular traffic through plasmodesmata, are clearly distinct from necks of mesophyll plasmodesmata. In contrast to the electrondense desmotubular area in mesophyll plasmodesmata, trichome plasmodesmata contain an electron-translucent circle in their center, surrounded by an electrondense ring. This latter ring is connected to the inner leaflet of the plasma membrane by multiple spokes or filaments. Two monoclonal antibodies raised against a maize plasmodesmal protein preparation (A. Turner et al., 1994, J Cell Sci. 107: 3351-3361) interact with both trichome and mesophyll N. clevelandii plasmodesmata. Based on the localization pattern and the high degree of cross-reactivity, both antibodies likely recognize a conserved structural component of plasmodesmata, and may be useful to mark plasmodesma in a variety of plants and tissues.
Cell Communication↗
Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells.
Agrobacterium genetically transforms plant cells by transferring a single-stranded DNA (ssDNA) copy of the transferred DNA (T-DNA) element, the T-strand, in a complex with Agrobacterium proteins VirD2, bound to the 5' end, and VirE2. VirE2 binds single-stranded nucleic acid cooperatively, fully coating the T-strand, and the protein localizes to the plant cell nucleus when transiently expressed. The coupling of ssDNA binding and nuclear localizing activities suggests that VirE2 alone could mediate nuclear localization of ssDNA. In this study, fluorescently labeled ssDNA accumulated in the plant cell nucleus specifically when microinjected as a complex with VirE2. Microinjected ssDNA alone remained cytoplasmic. Import of VirE2-ssDNA complex into the nucleus via a protein import pathway was supported by (i) the inhibition of VirE2-ssDNA complex import in the presence of wheat germ agglutinin or a nonhydrolyzable GTP analog, both known inhibitors of protein nuclear import, and (ii) the retardation of import when complexes were prepared from a VirE2 mutant impaired in ssDNA binding and nuclear import.
Bacterial Proteins↗
Three knotted1-like homeobox genes in Arabidopsis.
Five arabidopsis kn1-like homeobox genes were cloned through low-stringency screening of Arabidopsis cDNA libraries with the kn1 homeobox from maize. These five genes were named KNAT1-5 (for kn1-like Arabidopsis thaliana). An analysis of KNAT1 and 2 has been presented previously [19]. Here we present an analysis of the genes KNAT3, 4 and 5. On the basis of sequence and expression patterns, these three genes belong to the class II subfamily of kn1-like homeobox genes [16]. Low-stringency Southern analysis suggests several additional members of the class II genes exist in the Arabidopsis genome. The predicted amino acid sequences of the three genes share extensive homology outside of the homeodomain, including 84% between KNAT3 and KNAT4. Northern analysis shows that although all three genes are expressed in all tissues examined, the level of KNAT3 RNA is highest in young siliques, inflorescences and roots, KNAT4 RNA level is strongest in leaves and young siliques, and KNAT5 RNA level is highest in roots. The specificity of these patterns was confirmed by RNA fingerprint analysis. KNAT3 and 4 are light-regulated as they show reduced expression in etiolated seedlings and also in hy3, cop1 and det1 mutant backgrounds.
Amino Acid Sequence↗
Plasmodesmata: plant channels for molecules on the move.
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Biological Transport↗
Transfer of T-DNA from Agrobacterium to the plant cell.
Agrobacterium tumefaciens is the causative agent of crown gall, a disease of dicotyledonous plants characterized by a tumorous phenotype. Earlier in this century, scientific interest in A. tumefaciens was based on the possibility that the study of plant tumors might reveal mechanisms that were also operating in animal neoplasia. In the recent past, the tumorous growth was shown to result from the expression of genes coded for by a DNA segment of bacterial origin that was transferred and became stably integrated into the plant genome. This initial molecular characterization of the infection process suggested that Agrobacterium might be used to deliver genetic material into plants. The potential to genetically engineer plants generated renewed interest in the study of A. tumefaciens. In this review, we concentrate on the most recent advances in the study of Agrobacterium-mediated gene transfer, its relationship to conjugation, DNA processing and transport, and nuclear targeting. In the following discussion, references for earlier work can be found in more comprehensive reviews (Hooykaas and Schilperoort, 1992; Zambryski, 1992; Hooykaas and Beijersbergen, 1994).
Agrobacterium tumefaciens↗
Tobacco mosaic virus movement protein-mediated protein transport between trichome cells.
Tobacco mosaic virus movement protein (TMV MP) is required to mediate viral spread between plant cells via plasmodesmata. Plasmodesmata are cytoplasmic bridges that connect individual plant cells and ordinarily limit molecular diffusion to small molecules and metabolites with a molecular mass up to 1 kD. Here, we characterize functional properties of Nicotiana clevelandii trichome plasmodesmata and analyze their interaction with TMV MP. Trichomes constitute a linear cellular system and provide a predictable pathway of movement. Their plasmodesmata are functionally distinct from plasmodesmata in other plant cel types; they allow cell-to-cell diffusion of dextrans with a molecular mass up to 7 kD, and TMV MP does not increase this size exclusion limit for dextrans. In contrast, the 30-kD TMV MP itself moves between trichome cells and specifically mediates the translocation of a 90-kD beta-glucuronidase (GUS) reporter protein as a GUS::TMV MP fusion. Neither GUS by itself nor GUS in the presence of TMV MP moves between cells. These data imply that a plasmodesmal transport signal resides within TMV MP and is essential for movement. This signal confers selectivity to the translocated protein and cannot function in trans to support movement of other molecules.
Cloning, Molecular↗
Plasmodesmata. Gateways for rapid information transfer.
Viruses spread their genomes throughout infected plants by exploiting plasmodesmata, the cytoplasmic bridges that wire plant cells into a three-dimensional network and rapidly transport a variety of molecules.
Biological Transport↗
Nuclear import of Agrobacterium VirD2 and VirE2 proteins in maize and tobacco.
Previously, we have shown that Agrobacterium-plant cell transferred DNA (T-DNA) transport into the host cell nucleus is likely mediated by two specific bacterial proteins, VirD2 and VirE2. Here, we used these proteins to study molecular pathways of nuclear import. First, the role of VirE2 nuclear localization signals (NLSs) in the T-DNA transport pathway was examined by using tobacco plants transgenic for deletion mutants of VirE2. In these plants, the virulence of wild-type Agrobacterium was reduced possibly by competition for the cellular nuclear import machinery. Second, we analyzed the nuclear localization of VirE2 and VirD2 in the nonhost monocot maize. Part of the known recalcitrance of monocots to transformation by Agrobacterium could be due to a potential selectivity in nuclear import pathways in monocotyledonous and dicotyledonous plants. Nuclear transport of VirD2 and VirE2 in maize leaves and roots was compared to that in tobacco protoplasts and roots. Both proteins accumulated in maize leaf and tobacco protoplast nuclei as well as in nuclei of immature root cells. In contrast, VirD2 and VirE2 expressed in mature roots of maize and tobacco remained cytoplasmic. Point mutations of VirE2 nuclear localization signals, NSE 1 and NSE 2, also revealed that, in maize, the NSE 1 signal was mainly responsible for nuclear import; in contrast, both signals functioned independently in tobacco protoplasts.
Amino Acid Sequence↗
Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmal permeability.
Plasmodesmata are cytoplasmic bridges between plant cells thought to generally allow only the passage of small molecules and metabolites. However, large structures such as plant viruses also move from cell to cell via plasmodesmata. In tobacco mosaic virus (TMV) infection a viral movement protein (TMV-MP) mediates viral spread. Here, a microinjection assay is used to monitor the dynamics of TMV-MP function directly in wild-type plants. The results indicate that TMV-MP interacts with an endogenous plant pathway increasing plasmodesmal size exclusion limit to permit passage of 20-kDa dextrans. Furthermore, TMV-MP influences plasmodesmal size exclusion limit several cells distant from the injection site, indicating either that TMV-MP itself crosses plasmodesmata or that TMV-MP induces a diffusable signal capable of dilating microchannels of plasmodesmata. The region of TMV-MP responsible for increasing plasmodesmal size exclusion limit was mapped to the carboxyl-terminal part of the 268-amino acid residue protein between amino acid residues 126 and 224.
Biological Assay↗
Cell-to-cell movement of plant viruses.
To establish an infection, most plant viruses move from cell to cell in the plant. Virus-encoded movement proteins mediate this process and appear to use two mechanisms for transport. Both mechanisms involve interaction with and potential modification of plant intercellular connections, the plasmodesmata. Thus, although viral movement proteins are a diverse group, they share an ability to interact with specific plant components.
Movement↗