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The Agrobacterium growth pole ring (GPR) protein forms a hexameric ring at the growth pole (GP) that is essential for polar growth. GPR is large (2,115 amino acids) and contains 1,700 amino acids of continuous α-helices. To dissect potential GPR functional domains, we created deletions of regions with similarity to human apolipoprotein A-IV (396 amino acids), itself composed of α-helical domains. We also tested deletions of the GPR C terminus. Deletions were inducibly expressed as green fluorescent protein (GFP) fusion proteins and tested for merodiploid interference with wild-type (WT) GPR function, for partial function in cells lacking GPR, and for formation of paired fluorescent foci (indicative of hexameric rings) at the GP. Deletion of domains similar to human apolipoprotein A-IV in GPR caused defects in cell morphology when expressed in trans to WT GPR and provided only partial complementation to cells lacking GPR. Agrobacterium-specific domains A-IV-1 and A-IV-4 contain predicted coiled coil (CC) regions of 21 amino acids; deletion of CC regions produced severe defects in cell morphology in the interference assay. Mutants that produced the most severe effects on cell shape also failed to form paired polar foci. Modeling of A-IV-1 and A-IV-4 reveals significant similarity to the solved structure of human apolipoprotein A-IV. GPR C-terminal deletions profoundly blocked complementation. Finally, peptidoglycan (PG) synthesis is abnormally localized circumferentially in cells lacking GPR. The results support the hypothesis that GPR plays essential roles as an organizing center for membrane and PG synthesis during polar growth.IMPORTANCE Bacterial growth and division are extensively studied in model systems (Escherichia coli, Bacillus subtilis, and Caulobacter crescentus) that grow by dispersed insertion of new cell wall material along the length of the cell. An alternative growth mode-polar growth-is used by some Actinomycetales and Proteobacteria species. The latter phylum includes the family Rhizobiaceae, in which many species, including Agrobacterium tumefaciens, exhibit polar growth. Current research aims to identify growth pole (GP) factors. The Agrobacterium growth pole ring (GPR) protein is essential for polar growth and forms a striking hexameric ring structure at the GP. GPR is long (2,115 amino acids), and little is known about regions essential for structure or function. Genetic analyses demonstrate that the C terminus of GPR, and two internal regions with homology to human apolipoproteins (that sequester lipids), are essential for GPR function and localization to the GP. We hypothesize that GPR is an organizing center for membrane and cell wall synthesis during polar growth.

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.

The coordinated redistribution of sugars from mature "source" leaves to developing "sink" leaves requires tight regulation of sugar transport between cells via plasmodesmata (PD). Although fundamental to plant physiology, the mechanisms that control PD transport and thereby support development of new leaves have remained elusive. From a forward genetic screen for altered PD transport, we discovered that the conserved eukaryotic glucose-TOR (TARGET OF RAPAMYCIN) metabolic signaling network restricts PD transport in leaves. Genetic approaches and chemical or physiological treatments to either promote or disrupt TOR activity demonstrate that glucose-activated TOR decreases PD transport in leaves. We further found that TOR is significantly more active in mature leaves photosynthesizing excess sugars than in young, growing leaves, and that this increase in TOR activity correlates with decreased rates of PD transport. We conclude that leaf cells regulate PD trafficking in response to changing carbohydrate availability monitored by the TOR pathway.

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.

Agrobacterium tumefaciens grows by addition of peptidoglycan (PG) at one pole of the bacterium. During the cell cycle, the cell needs to maintain two different developmental programs, one at the growth pole and another at the inert old pole. Proteins involved in this process are not yet well characterized. To further characterize the role of pole-organizing protein A. tumefaciens PopZ (PopZ At ), we created deletions of the five PopZ At domains and assayed their localization. In addition, we created a popZAt deletion strain (ΔpopZAt ) that exhibited growth and cell division defects with ectopic growth poles and minicells, but the strain is unstable. To overcome the genetic instability, we created an inducible PopZ At strain by replacing the native ribosome binding site with a riboswitch. Cultivated in a medium without the inducer theophylline, the cells look like ΔpopZAt cells, with a branching and minicell phenotype. Adding theophylline restores the wild-type (WT) cell shape. Localization experiments in the depleted strain showed that the domain enriched in proline, aspartate, and glutamate likely functions in growth pole targeting. Helical domains H3 and H4 together also mediate polar localization, but only in the presence of the WT protein, suggesting that the H3 and H4 domains multimerize with WT PopZ At , to stabilize growth pole accumulation of PopZ AtIMPORTANCEAgrobacterium tumefaciens is a rod-shaped bacterium that grows by addition of PG at only one pole. The factors involved in maintaining cell asymmetry during the cell cycle with an inert old pole and a growing new pole are not well understood. Here we investigate the role of PopZ At , a homologue of Caulobacter crescentus PopZ (PopZ Cc ), a protein essential in many aspects of pole identity in C. crescentus We report that the loss of PopZ At leads to the appearance of branching cells, minicells, and overall growth defects. As many plant and animal pathogens also employ polar growth, understanding this process in A. tumefaciens may lead to the development of new strategies to prevent the proliferation of these pathogens. In addition, studies of A. tumefaciens will provide new insights into the evolution of the genetic networks that regulate bacterial polar growth and cell division.

Agrobacterium tumefaciens grows by addition of peptidoglycan (PG) at one pole of the bacterium. During the cell cycle, the cell needs to maintain two different developmental programs, one at the growth pole and another at the inert old pole. Proteins involved in this process are not yet well characterized. To further characterize the role of pole-organizing protein A. tumefaciens PopZ (PopZ At ), we created deletions of the five PopZ At domains and assayed their localization. In addition, we created a popZAt deletion strain (ΔpopZAt ) that exhibited growth and cell division defects with ectopic growth poles and minicells, but the strain is unstable. To overcome the genetic instability, we created an inducible PopZ At strain by replacing the native ribosome binding site with a riboswitch. Cultivated in a medium without the inducer theophylline, the cells look like ΔpopZAt cells, with a branching and minicell phenotype. Adding theophylline restores the wild-type (WT) cell shape. Localization experiments in the depleted strain showed that the domain enriched in proline, aspartate, and glutamate likely functions in growth pole targeting. Helical domains H3 and H4 together also mediate polar localization, but only in the presence of the WT protein, suggesting that the H3 and H4 domains multimerize with WT PopZ At , to stabilize growth pole accumulation of PopZ AtIMPORTANCEAgrobacterium tumefaciens is a rod-shaped bacterium that grows by addition of PG at only one pole. The factors involved in maintaining cell asymmetry during the cell cycle with an inert old pole and a growing new pole are not well understood. Here we investigate the role of PopZ At , a homologue of Caulobacter crescentus PopZ (PopZ Cc ), a protein essential in many aspects of pole identity in C. crescentus We report that the loss of PopZ At leads to the appearance of branching cells, minicells, and overall growth defects. As many plant and animal pathogens also employ polar growth, understanding this process in A. tumefaciens may lead to the development of new strategies to prevent the proliferation of these pathogens. In addition, studies of A. tumefaciens will provide new insights into the evolution of the genetic networks that regulate bacterial polar growth and cell division.

UnlabelledAgrobacterium tumefaciens is a rod-shaped Gram-negative bacterium that elongates by unipolar addition of new cell envelope material. Approaching cell division, the growth pole transitions to a nongrowing old pole, and the division site creates new growth poles in sibling cells. The A. tumefaciens homolog of the Caulobacter crescentus polar organizing protein PopZ localizes specifically to growth poles. In contrast, the A. tumefaciens homolog of the C. crescentus polar organelle development protein PodJ localizes to the old pole early in the cell cycle and accumulates at the growth pole as the cell cycle proceeds. FtsA and FtsZ also localize to the growth pole for most of the cell cycle prior to Z-ring formation. To further characterize the function of polar localizing proteins, we created a deletion of A. tumefaciens podJ (podJAt). ΔpodJAt cells display ectopic growth poles (branching), growth poles that fail to transition to an old pole, and elongated cells that fail to divide. In ΔpodJAt cells, A. tumefaciens PopZ-green fluorescent protein (PopZAt-GFP) persists at nontransitioning growth poles postdivision and also localizes to ectopic growth poles, as expected for a growth-pole-specific factor. Even though GFP-PodJAt does not localize to the midcell in the wild type, deletion of podJAt impacts localization, stability, and function of Z-rings as assayed by localization of FtsA-GFP and FtsZ-GFP. Z-ring defects are further evidenced by minicell production. Together, these data indicate that PodJAt is a critical factor for polar growth and that ΔpodJAt cells display a cell division phenotype, likely because the growth pole cannot transition to an old pole.ImportanceHow rod-shaped prokaryotes develop and maintain shape is complicated by the fact that at least two distinct species-specific growth modes exist: uniform sidewall insertion of cell envelope material, characterized in model organisms such as Escherichia coli, and unipolar growth, which occurs in several alphaproteobacteria, including Agrobacterium tumefaciens Essential components for unipolar growth are largely uncharacterized, and the mechanism constraining growth to one pole of a wild-type cell is unknown. Here, we report that the deletion of a polar development gene, podJAt, results in cells exhibiting ectopic polar growth, including multiple growth poles and aberrant localization of cell division and polar growth-associated proteins. These data suggest that PodJAt is a critical factor in normal polar growth and impacts cell division in A. tumefaciens.

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Agrobacterium tumefaciens elongates by addition of peptidoglycan (PG) only at the pole created by cell division, the growth pole, whereas the opposite pole, the old pole, is inactive for PG synthesis. How Agrobacterium assigns and maintains pole asymmetry is not understood. Here, we investigated whether polar growth is correlated with novel pole-specific localization of proteins implicated in a variety of growth and cell division pathways. The cell cycle of A. tumefaciens was monitored by time-lapse and superresolution microscopy to image the localization of A. tumefaciens homologs of proteins involved in cell division, PG synthesis and pole identity. FtsZ and FtsA accumulate at the growth pole during elongation, and improved imaging reveals FtsZ disappears from the growth pole and accumulates at the midcell before FtsA. The L,D-transpeptidase Atu0845 was detected mainly at the growth pole. A. tumefaciens specific pole-organizing protein (Pop) PopZAt and polar organelle development (Pod) protein PodJAt exhibited dynamic yet distinct behavior. PopZAt was found exclusively at the growing pole and quickly switches to the new growth poles of both siblings immediately after septation. PodJAt is initially at the old pole but then also accumulates at the growth pole as the cell cycle progresses suggesting that PodJAt may mediate the transition of the growth pole to an old pole. Thus, PopZAt is a marker for growth pole identity, whereas PodJAt identifies the old pole.

A fundamental mystery of plant cell biology is the occurrence of "stromules," stroma-filled tubular extensions from plastids (such as chloroplasts) that are universally observed in plants but whose functions are, in effect, completely unknown. One prevalent hypothesis is that stromules exchange signals or metabolites between plastids and other subcellular compartments, and that stromules are induced during stress. Until now, no signaling mechanisms originating within the plastid have been identified that regulate stromule activity, a critical missing link in this hypothesis. Using confocal and superresolution 3D microscopy, we have shown that stromules form in response to light-sensitive redox signals within the chloroplast. Stromule frequency increased during the day or after treatment with chemicals that produce reactive oxygen species specifically in the chloroplast. Silencing expression of the chloroplast NADPH-dependent thioredoxin reductase, a central hub in chloroplast redox signaling pathways, increased chloroplast stromule frequency, whereas silencing expression of nuclear genes related to plastid genome expression and tetrapyrrole biosynthesis had no impact on stromules. Leucoplasts, which are not photosynthetic, also made more stromules in the daytime. Leucoplasts did not respond to the same redox signaling pathway but instead increased stromule formation when exposed to sucrose, a major product of photosynthesis, although sucrose has no impact on chloroplast stromule frequency. Thus, different types of plastids make stromules in response to distinct signals. Finally, isolated chloroplasts could make stromules independently after extraction from the cytoplasm, suggesting that chloroplast-associated factors are sufficient to generate stromules. These discoveries demonstrate that chloroplasts are remarkably autonomous organelles that alter their stromule frequency in reaction to internal signal transduction pathways.

UnlabelledThe synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as Escherichia coli that grow by dispersed lateral insertion of PG, little is known of the processes that direct polar PG synthesis in other bacteria such as the Rhizobiales. To better understand polar growth in the Rhizobiales Agrobacterium tumefaciens, we first surveyed its genome to identify homologs of (~70) well-known PG synthesis components. Since most of the canonical cell elongation components are absent from A. tumefaciens, we made fluorescent protein fusions to other putative PG synthesis components to assay their subcellular localization patterns. The cell division scaffolds FtsZ and FtsA, PBP1a, and a Rhizobiales- and Rhodobacterales-specific l,d-transpeptidase (LDT) all associate with the elongating cell pole. All four proteins also localize to the septum during cell division. Examination of the dimensions of growing cells revealed that new cell compartments gradually increase in width as they grow in length. This increase in cell width is coincident with an expanded region of LDT-mediated PG synthesis activity, as measured directly through incorporation of exogenous d-amino acids. Thus, unipolar growth in the Rhizobiales is surprisingly dynamic and represents a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli.ImportanceMany rod-shaped bacteria, including pathogens such as Brucella and Mycobacteriu, grow by adding new material to their cell poles, and yet the proteins and mechanisms contributing to this process are not yet well defined. The polarly growing plant pathogen Agrobacterium tumefaciens was used as a model bacterium to explore these polar growth mechanisms. The results obtained indicate that polar growth in this organism is facilitated by repurposed cell division components and an otherwise obscure class of alternative peptidoglycan transpeptidases (l,d-transpeptidases). This growth results in dynamically changing cell widths as the poles expand to maturity and contrasts with the tightly regulated cell widths characteristic of canonical rod-shaped growth. Furthermore, the abundance and/or activity of l,d-transpeptidases appears to associate with polar growth strategies, suggesting that these enzymes may serve as attractive targets for specifically inhibiting growth of Rhizobiales, Actinomycetales, and other polarly growing bacterial pathogens.

The accessory Sec system of Streptococcus gordonii is comprised of SecY2, SecA2, and five proteins (Asp1 through -5) that are required for the export of a serine-rich glycoprotein, GspB. We have previously shown that a number of the Asps interact with GspB, SecA2, or each other. To further define the roles of these Asps in export, we examined their subcellular localization in S. gordonii and in Escherichia coli expressing the streptococcal accessory Sec system. In particular, we assessed how the locations of these accessory Sec proteins were altered by the presence of other components. Using fluorescence microscopy, we found in E. coli that SecA2 localized within multiple foci at the cell membrane, regardless of whether other accessory Sec proteins were expressed. Asp2 alone localized to the cell poles but formed a similar punctate pattern at the membrane when SecA2 was present. Asp1 and Asp3 localized diffusely in the cytosol when expressed alone or with SecA2. However, these proteins redistributed to the membrane in a punctate arrangement when all of the accessory Sec components were present. Cell fractionation studies with S. gordonii further corroborated these microscopy results. Collectively, these findings indicate that Asp1 to -3 are not integral membrane proteins that form structural parts of the translocation channel. Instead, SecA2 serves as a docking site for Asp2, which in turn attracts a complex of Asp1 and Asp3 to the membrane. These protein interactions may be important for the trafficking of GspB to the cell membrane and its subsequent translocation.

Plasmodesmata (PD) structure and function vary temporally and spatially during all stages of plant development. PD that originate during, or post, cell division are designated as primary or secondary according to classical terminology. PD structure may be simple, twinned, or branched. Studies of PD during leaf, root, and embryo development have lead to the generalization that cells in less mature tissues contain predominantly simple PD. New quantitative analyses reveal that twinned and branched PD also occur in immature tissues. New data also highlight the versatility of viral movement proteins as tags for labeling PD in immature tissues as well as PD in mature tissues. A summary of the formation and function of primary, secondary, and branched PD during leaf, trichome, embryo, apical meristem, vascular cambium, and root development underscores the remarkable and indispensible plant-specific intercellular communication system that is mediated by PD.

Genetic and physiological analyses implicate auxin flux in patterning, initiation and growth of floral organs. Within the Arabidopsis flower, the ETTIN/ARF3 transcription factor responds to auxin to effect perianth organ number and reproductive organ differentiation. This work describes a modifier of ettin that causes filamentous, mispositioned outer whorl organs and reduced numbers of malformed stamens in the double mutant. The modifier was discovered to be a new allele of the seuss (seu) mutant. SEU encodes a novel protein that is predicted to transcriptionally co-repress the AGAMOUS floral organ identity gene. The effects of seu on ett are shown to be independent of the SEU-AG pathway. Furthermore, morphological, physiological and genetic evidenceimplicate SEU in auxin-regulated growth and development. seu has a pleiotropic phenotype that includes reductions in several classic auxin responses such as apical dominance, lateral root initiation, sensitivity to exogenous auxin and activation of the DR5 auxin response reporter. seu displays a synergistic interaction with the auxin response mutant pinoid, producing flowers with few outer whorl organs. Collectively, these data suggest that SEU is a novel factor affecting auxin response. A model is proposed in which SEU functions jointly with ETT in auxin response to promote floral organ patterning and growth.

Fundamental research aimed to determine the limits of the Agrobacterium transfer DNA (T-DNA) element that stably inserted into plant nuclear DNA to cause crown gall tumor formation. The T-DNA borders were discovered to be exceedingly precise, revealing that T-DNA insertion into the plant genome was reproducible and exact. Deletion of the internal regions of the T-DNA, to remove the tumor forming genes, while retaining the T-DNA borders, resulted again in efficient DNA transfer to plant cells, but now such cells were capable of completely normal growth and differentiation. Thus, the internal region of the T-DNA was not needed for DNA transfer, and one could envisage insertion of any DNA of interest in between the T-DNA borders. Thus began plant genetic engineering. [ABSTRACT FROM AUTHOR]