Segregation of four Agrobacterium tumefaciens replicons during polar growth: PopZ and PodJ control segregation of essential replicons.

Agrobacterium tumefaciens C58 contains four replicons, circular chromosome (CC), linear chromosome (LC), cryptic plasmid (pAt), and tumor-inducing plasmid (pTi), and grows by polar growth from a single growth pole (GP), while the old cell compartment and its old pole (OP) do not elongate. We monitored the replication and segregation of these four genetic elements during polar growth. The three largest replicons (CC, LC, pAt) reside in the OP compartment prior to replication; post replication one copy migrates to the GP prior to division. CC resides at a fixed location at the OP and replicates first. LC does not stay fixed at the OP once the cell cycle begins and replicates from varied locations 20 min later than CC. pAt localizes similarly to LC prior to replication, but replicates before the LC and after the CC. pTi does not have a fixed location, and post replication it segregates randomly throughout old and new cell compartments, while undergoing one to three rounds of replication during a single cell cycle. Segregation of the CC and LC is dependent on the GP and OP identity factors PopZ and PodJ, respectively. Without PopZ, replicated CC and LC do not efficiently partition, resulting in sibling cells without CC or LC. Without PodJ, the CC and LC exhibit abnormal localization to the GP at the beginning of the cell cycle and replicate from this position. These data reveal PodJ plays an essential role in CC and LC tethering to the OP during early stages of polar growth.

GROWTH POLE RING protein forms a 200-nm-diameter ring structure essential for polar growth and rod shape in Agrobacterium tumefaciens.

Polar growth in Agrobacterium pirates and repurposes well-known bacterial cell cycle proteins, such as FtsZ, FtsA, PopZ, and PodJ. Here we identify a heretofore unknown protein that we name GROWTH POLE RING (GPR) due to its striking localization as a hexameric ring at the growth pole during polar growth. GPR also localizes at the midcell late in the cell cycle just before division, where it is then poised to be precisely localized at new growth poles in sibling cells. GPR is 2,115 aa long, with two N-terminal transmembrane domains placing the bulk of the protein in the cytoplasm, N- and C-terminal proline-rich disordered regions, and a large 1,700-aa central region of continuous α-helical domains. This latter region contains 12 predicted adjacent or overlapping apolipoprotein domains that may function to sequester lipids during polar growth. Stable genetic deletion or riboswitch-controlled depletion results in spherical cells that grow poorly; thus, GPR is essential for wild-type growth and morphology. As GPR has no predicted enzymatic domains and it forms a distinct 200-nm-diameter ring, we propose that GPR is a structural component of an organizing center for peptidoglycan and membrane syntheses critical for cell envelope formation during polar growth. GPR homologs are found in numerous Rhizobiales; thus, our results and proposed model are fundamental to understanding polar growth strategy in a variety of bacterial species.

Cell growth: the power of symplastic isolation.

Cotton (Gossypium hirsutum) fibers are single-celled seed coat hairs that elongate up to 2mm per day during a phase of rapid growth. Recent evidence suggests this growth is orchestrated by a series of events which includes temporary closure of plasmodesmata.

Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states.

Plant cells rely on plasmodesmata for intercellular transport of small signaling molecules as well as larger informational macromolecules such as proteins. A green fluorescent protein (GFP) reporter and low-pressure microprojectile bombardment were used to quantify the degree of symplastic continuity between cells of the leaf at different developmental stages and under different growth conditions. Plasmodesmata were observed to be closed to the transport of GFP or dilated to allow the traffic of GFP. In sink leaves, between 34% and 67% of the cells transport GFP (27 kD), and between 30% and 46% of the cells transport double GFP (54 kD). In leaves in transition transport was reduced; between 21% and 46% and between 2% and 9% of cells transport single and double GFP, respectively. Thus, leaf age dramatically affects the ability of cells to exchange proteins nonselectively. Further, the number of cells allowing GFP or double GFP movement was sensitive to growth conditions because greenhouse-grown plants exhibited higher diffusion rates than culture-grown plants. These studies reveal that leaf cell plasmodesmata are dynamic and do not have a set size exclusion limit. We also examined targeted movement of the movement protein of tobacco mosaic virus fused to GFP, P30::GFP. This 58-kD fusion protein localizes to plasmodesmata, consistently transits from up to 78% of transfected cells, and was not sensitive to developmental age or growth conditions. The relative number of cells containing dilated plasmodesmata varies between different species of tobacco, with Nicotiana clevelandii exhibiting greater diffusion of proteins than Nicotiana tabacum.

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.

Subcellular localization determines the availability of non-targeted proteins to plasmodesmatal transport.

BACKGROUND: Individual plant cells are encased in a cell wall. To enable cell-to-cell communication, plants have evolved channels, termed plasmodesmata, to span thick walls and interconnect the cytoplasm between adjacent cells. How macromolecules pass through these channels is now beginning to be understood. RESULTS: Using two green fluorescent protein (GFP) reporters and a non-invasive transfection system, we assayed for intercellular macromolecular traffic in leaf epidermal cells. Plasmodesmata were found in different states of dilation. We could distinguish two forms of protein movement across plasmodesmata, non-targeted and targeted. Although leaves have generally been considered closed to non-specific transport of macromolecules, we found that 23% of the cells had plasmodesmatal channels in a dilated state, allowing GFP that was not targeted to plasmodesmata to move into neighboring cells. GFP fusions that were targeted to the cytoskeleton or to the endoplasmic reticulum did not move between cells, whereas those that were localized to the cytoplasm or nucleus diffused to neighboring cells in a size-dependent manner. Superimposed upon this non-specific exchange, proteins that were targeted to the plasmodesmata could transit efficiently between 62% of transfected cells. CONCLUSIONS: A significant population of leaf cells contain plasmodesmata in a dilated state, allowing macromolecular transport between cells. Protein movement potential is regulated by subcellular address and size. These parameters of protein movement illustrate how gradients of signaling macromolecules could be formed and regulated, and suggest that non-cell-autonomous development in plants may be more significant than previously assumed.

Cell-to-cell and systemic movement of recombinant green fluorescent protein-tagged turnip crinkle viruses.

To facilitate analyses of turnip crinkle virus (TCV) cell-to-cell and systemic movement, we created a series of recombinant viruses expressing green fluorescent protein (GFP) either as substitutions of coat protein (CP) sequences or as fusions to movement proteins (MPs). Constructs were used to inoculate leaves of Arabidopsis seedlings. TCV carrying its two native MPs and GFP fused near the start of CP translation (GFP DeltaCP) resulted in cell-to-cell movement manifested by the expansion of fluorescent foci on inoculated leaves. GFP fusions to either MP were inactive for movement. However, TCV carrying the p9-GFP fusion, which expresses a functional p8 gene, could be complemented for cell-to-cell movement by coinoculation with virus carrying native p9 but mutant for p8. This same coinoculation combination also lead to systemic spread of GFP fluorescence to noninoculated leaves, as the complementing virus carries native CP. Complementation for systemic movement of virus carrying GFP DeltaCP constructs was achieved by inoculation onto transgenic plants expressing TCV CP. GFP-tagged TCV movement was detected throughout the plant, including the inflorescence stem, cauline leaves, flowers, siliques, and substructures such as organ primordia and meristematic regions. The recombinant viruses described herein provide (1) genetic information relevant to define regions of TCV that can, or cannot, be manipulated by insertion of foreign coding sequences and (2) a set of tools to allow the study of viral cell-to-cell and long-distance movement in the model plant system Arabidopsis.

Nuclear localization of turnip crinkle virus movement protein p8.

Turnip crinkle virus (TCV) is a single-stranded positive-sense RNA virus of the Carmovirus genus. Two of its five open reading frames (ORFs), encoding proteins of 8 and 9 kDa, are required for cell-to-cell movement of the virus. These movement proteins (MPs) were fused to green fluorescent protein (GFP) to determine their cellular localization. In protoplasts, p9-GFP, like GFP itself, is found throughout the cytoplasm, as well as in cell nuclei. In contrast, p8-GFP was confined to the cell nucleus. Similar localization patterns were observed when specific small peptide epitopes were fused to p8 and p9 proteins instead of GFP. The cytoplasmic localization of p9-GFP and nuclear localization of p8-GFP were also detected in leaves after particle bombardment of DNA encoding these fusion proteins or after overexpression of p8-GFP in transgenic Arabidopsis seedlings. The expression of the GFP fusion proteins by recombinant TCV viruses in infected protoplasts or on inoculated Arabidopsis leaves produced similar patterns. Unlike TMV-MP and other MPs studied to date, no obvious punctuate expression in the cell wall or association with the cytoskeleton was detected. The sequence analysis of p8 revealed two unique nuclear localization signals (NLSs), which were not conserved within p8 homologues of other viruses in the genus Carmovirus. Mutation in either of these NLSs did not disrupt the nuclear localization of p8-GFP. However, when both NLSs were mutated, p8-GFP expression was no longer restricted to cell nuclei. The NLSs are not required for cell-to-cell movement; TCV recombinant viruses mutated in one or both NLSs could still facilitate cell-to-cell movement of the virus. The nuclear localization of p8 suggests a novel function for this protein in the cell nucleus.

Auxin and ETTIN in Arabidopsis gynoecium morphogenesis.

The phytohormone auxin has wide-ranging effects on growth and development. Genetic and physiological approaches implicate auxin flux in determination of floral organ number and patterning. This study uses a novel technique of transiently applying a polar auxin transport inhibitor, N-1-naphthylphthalamic acid (NPA), to developing Arabidopsis flowers to further characterize the role of auxin in organogenesis. NPA has marked effects on floral organ number as well as on regional specification in wild-type gynoecia, as defined by morphological and histological landmarks for regional boundaries, as well as tissue-specific reporter lines. NPA's effects on gynoecium patterning mimic the phenotype of mutations in ETTIN, a member of the auxin response factor family of transcription factors. In addition, application of different concentrations of NPA reveal an increased sensitivity of weak ettin alleles to disruptions in polar auxin transport. In contrast, the defects found in spatula gynoecia are partially rescued by treatment with NPA. A model is proposed suggesting an apical-basal gradient of auxin during gynoecium development. This model provides a mechanism linking ETTIN's putative transcriptional regulation of auxin-responsive genes to the establishment or elaboration of tissue patterning during gynoecial development.

VirB6 is required for stabilization of VirB5 and VirB3 and formation of VirB7 homodimers in Agrobacterium tumefaciens.

VirB6 from Agrobacterium tumefaciens is an essential component of the type IV secretion machinery for T pilus formation and genetic transformation of plants. Due to its predicted topology as a polytopic inner membrane protein, it was proposed to form the transport pore for cell-to-cell transfer of genetic material and proteinaceous virulence factors. Here, we show that the absence of VirB6 leads to reduced cellular levels of VirB5 and VirB3, which were proposed to assist T pilus formation as minor component(s) or assembly factor(s), respectively. Overexpression of virB6 in trans restored levels of cell-bound and T pilus-associated VirB5 to wild type but did not restore VirB3 levels. Thus, VirB6 has a stabilizing effect on VirB5 accumulation, thereby regulating T pilus assembly. In the absence of VirB6, cell-bound VirB7 monomers and VirB7-VirB9 heterodimers were reduced and VirB7 homodimer formation was abolished. This effect could not be restored by expression of VirB6 in trans. Expression of TraD, a component of the transfer machinery of the IncN plasmid pKM101, with significant sequence similarity to VirB6, restored neither protein levels nor bacterial virulence but partly permitted T pilus formation in a virB6 deletion strain. VirB6 may therefore regulate T pilus formation by direct interaction with VirB5, and wild-type levels of VirB3 and VirB7 homodimers are not required.

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.

New tools for protein linkage mapping and general two-hybrid screening.

The two-hybrid system has proved to be a facile method for detecting and analyzing protein-protein interactions. An expanded application of this system, protein linkage mapping, provides a means of identifying interactions on a global scale and should prove a powerful tool in analyzing whole genomes as their sequences become available. To overcome some of the inherent difficulties in such a large-scale approach, we have constructed a set of new strains and vectors that will allow for more efficient screening. The strains contain a GAL1-URA3 reporter for positive and negative selection, as well as a UAS(G)-lacZ reporter. The strains are of opposite mating types, permitting libraries present in one strain to be easily screened against a second library in the companion strain. We also constructed a family of CEN-based vectors for expression of both Gal4 DNA-binding and activation domain fusions. These plasmids include a hemagglutinin epitope tag and different polylinkers to increase the ease of subcloning. CEN-based vectors are maintained at 1-2 copies per cell, limiting the number of individual cells containing multiple plasmids that can confuse further analyses, and ensuring that fusions are not expressed at toxic levels. Using these vectors, both homo- and heterodimeric interactions resulted in up to 10-fold higher reporter gene transcription than obtained with 2micro;-based plasmids, despite significantly lower protein levels. In addition to protein linkage mapping, these reagents should be generally useful in standard two-hybrid applications.

Vir proteins stabilize VirB5 and mediate its association with the T pilus of Agrobacterium tumefaciens.

Three VirB proteins (VirB1*, VirB2, and VirB5) have been implicated as putative components of the T pilus from Agrobacterium tumefaciens, which likely mediates binding to plant cells followed by transfer of genetic material. Recently, VirB2 was indeed shown to be its major component (E.-M. Lai and C. I. Kado, J. Bacteriol. 180:2711-2717, 1998). Here, the influence of other Vir proteins on the stability and cellular localization of VirB1*, VirB2, and VirB5 was analyzed. Solubility of VirB1* and membrane association of VirB2 proved to be inherent features of these proteins, independent of virulence gene induction. In contrast, cellular levels of VirB5 were strongly reduced in the absence of other Vir proteins, indicating its stabilization by protein-protein interactions. The assembly and composition of the T pilus were analyzed in nopaline strain C58(pTiC58), its flagellum-free derivative NT1REB(pJK270), and octopine strain A348(pTiA6) following optimized virulence gene induction on solid agar medium. In all strains VirB2 was the major pilus component and VirB5 cofractionated during several purification steps, such as ultracentrifugation, gel filtration, and sucrose gradient centrifugation. VirB5 may therefore be directly involved in pilus assembly, possibly as minor component. In contrast, secreted VirB1* showed no association with the T pilus. In-frame deletions in genes virB1, virB2, virB5, and virB6 blocked the formation of virulence gene-dependent extracellular high-molecular-weight structures. Thus, an intact VirB machinery as well as VirB2 and VirB5 are required for T-pilus formation.

Plasmodesmata signaling: many roles, sophisticated statutes.

Recent advances have increased our understanding of plasmodesmata function, their architecture as it relates to signaling capacity, the temporal and spatial regulation of their permeability, and their roles in systemic transport of macromolecules, non-cell autonomous development, and, potentially, plant defense.

Phloem transport: Are you chaperoned?

Long-distance transport via the vasculature in plants is critical for nutrient dissemination, as well as transport of growth regulatory molecules such as hormones. Evidence is now accumulating that protein and RNA molecules also use this transport pathway, possibly to regulate developmental and physiological processes.

Temporal and spatial regulation of symplastic trafficking during development in Arabidopsis thaliana apices.

Plasmodesmata provide symplastic continuity linking individual plant cells. However, specialized cells may be isolated, either by the absence of plasmodesmata or by down regulation of the cytoplasmic flux through these channels, resulting in the formation of symplastic domains. Maintenance of these domains may be essential for the co-ordination of growth and development. While cells in the center of the meristem divide slowly and remain undifferentiated, cells on the meristem periphery divide more frequently and respond to signals determining organ fate. Such symplastic domains were visualized within shoot apices of Arabidopsis, by monitoring fluorescent symplastic tracers (HPTS: 8-hydroxypyrene 1,3,6 trisulfonic acid and CF: carboxy fluorescein). Tracers were loaded through cut leaves and distributed throughout the whole plant. Confocal laser scanning microscopy on living Arabidopsis plants indicates that HPTS moves via the vascular tissue from leaves to the apex where the tracer exits the phloem and moves symplastically into surrounding cells. The distribution of HPTS was monitored in vegetative apices, and just prior to, during, and after the switch to production of flowers. The apices of vegetative plants loaded with HPTS had detectable amounts of tracer in the tunica layer of the meristem and in very young primordia, whereas the corpus of the meristem excluded tracer uptake. Fluorescence signal intensity decreased prior to the onset of flowering. Moreover, at approximately the time the plants were committed to flowering, HPTS was undetectable in the inflorescence meristem or young primordia. Later in development, after several secondary inflorescences and mature siliques appeared, inflorescence apices again showed tracer loading at levels comparable to that of vegetative apices. Thus, analysis of fluorescent tracer movement via plasmodesmata reveals there is distinct temporal and spatial regulation of symplastic domains at the apex, dependent on the developmental stage of the plant.

Photoinduction of flower identity in vegetatively biased primordia.

Far-red light and long photoperiods promote flowering in Arabidopsis. We report here that when 30-day-old vegetative plants were induced with a continuous light treatment enriched in far-red light, flowers developed directly from previously initiated primordia. Specifically, plants induced with our continuous incandescent-enriched (CI) treatment produced an average of two primary-axis nodes with a leaf/flower phenotype, indicating that approximately two leaf/paraclade primordia per plant produced an individual flower from tissue that typically would differentiate into a paraclade (secondary inflorescence). Assays for APETALA1::beta-glucuronidase activity during the CI photoinduction treatment indicated that the floral meristem identity gene APETALA1 was transcriptionally activated in primordia with a leaf/paraclade bias and in primordia committed to leaf/paraclade development. APETALA1::beta-glucuronidase activity levels were initially highest in young primordia but were not correlated strictly with primordium fate. These results indicate that primordium fate can be modified after primordium initiation and that developing primordia respond quantitatively to floral induction signals.