PopZ identifies the new pole, and PodJ identifies the old pole during polar growth in Agrobacterium tumefaciens.

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.

Chloroplasts extend stromules independently and in response to internal redox signals.

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.

Loss of PodJ in Agrobacterium tumefaciens Leads to Ectopic Polar Growth, Branching, and Reduced Cell Division.

UNLABELLED: Agrobacterium 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. IMPORTANCE: How 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.

Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens.

Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes.

Plasmodesmata formation and cell-to-cell transport are reduced in decreased size exclusion limit 1 during embryogenesis in Arabidopsis.

In plants, plasmodesmata (PD) serve as channels for micromolecular and macromolecular cell-to-cell transport. Based on structure, PD in immature tissues are classified into two types, simple and branched (X- and Y-shaped) or twinned. The maximum size of molecules capable of PD transport defines PD aperture, known as the PD size exclusion limit. Here we report an Arabidopsis mutation, decreased size exclusion limit1 (dse1), that exhibits reduced cell-to-cell transport of the small (524 Da) fluorescent tracer 8-hydroxypyrene-1,3,6-trisulfonic acid at the midtorpedo stage of embryogenesis. Correspondingly, the fraction of X- and Y-shaped and twinned PD was reduced in dse1 embryos compared with WT embryos at this stage, suggesting that the frequency of PD is related to transport capability. dse1 is caused by a point mutation in At4g29860 (previously termed TANMEI) at the last donor splice site of its transcript, resulting in alternative splicing in both the first intron and the last intron. AtDSE1 is a conserved eukaryotic 386-aa WD-repeat protein critical for Arabidopsis morphogenesis and reproduction. Similar to its homologs in mouse, null mutants are embryo-lethal. The weak loss-of-function mutant dse1 exhibits pleiotropic phenotypes, including retarded vegetative growth, delayed flowering time, dysfunctional male and female organs, and delayed senescence. Finally, silencing of DSE1 in Nicotiana benthamiana leaves leads to reduced movement of GFP fused to tobacco mosaic virus movement protein. Thus, DSE1 is important for regulating PD transport between plant cells.

Organ boundary1 defines a gene expressed at the junction between the shoot apical meristem and lateral organs.

We identify a gene, organ boundary1 (OBO1), by its unique pattern of enhancer- driven GFP expression at the boundaries between the apical meristems and lateral organs in Arabidopsis embryos, seedlings, and mature plants. OBO1 also is expressed at the root apical meristem and in distinct cell files surrounding this area. OBO1 is one of a 10-member plant-specific gene family encoding a single small domain (133 amino acids) with unknown function. One member of this gene family, OBO2, is identical to a previously studied gene, light-sensitive hypocotyl1. Overexpression of OBO1 causes an abnormal number and size of petals and petal-stamen fusions. The patterns of OBO1 gene expression are distinct but overlap with other genes involved in boundary formation in the Arabidopsis shoot apical meristem, including cup-shaped cotyledon, lateral organ boundaries, blade-on-petiole, asymmetric leaves, and lateral organ fusion. Nuclear localization of OBO1 suggests that it might act with one or more of the transcription factors encoded by the foregoing genes. Ablation of the specific cells expressing OBO1 leads to loss of the shoot apical meristem and lateral organs. Thus, the cells expressing OBO1 are important for meristem maintenance and organogenesis in Arabidopsis.

Organelle-nucleus cross-talk regulates plant intercellular communication via plasmodesmata.

We use Arabidopsis thaliana embryogenesis as a model system for studying intercellular transport via plasmodesmata (PD). A forward genetic screen for altered PD transport identified increased size exclusion limit (ise) 1 and ise2 mutants with increased intercellular transport of fluorescent 10-kDa tracers. Both ise1 and ise2 exhibit increased formation of twinned and branched PD. ISE1 encodes a mitochondrial DEAD-box RNA helicase, whereas ISE2 encodes a DEVH-type RNA helicase. Here, we show that ISE2 foci are localized to the chloroplast stroma. Surprisingly, plastid development is defective in both ise1 and ise2 mutant embryos. In an effort to understand how RNA helicases that localize to different organelles have similar impacts on plastid and PD development/function, we performed whole-genome expression analyses. The most significantly affected class of transcripts in both mutants encode products that target to and enable plastid function. These results reinforce the importance of plastid-mitochondria-nucleus cross-talk, add PD as a critical player in the plant cell communication network, and thereby illuminate a previously undescribed signaling pathway dubbed organelle-nucleus-plasmodesmata signaling. Several genes with roles in cell wall synthesis and modification are also differentially expressed in both mutants, providing new targets for investigating PD development and function.

Differential localization of the streptococcal accessory sec components and implications for substrate export.

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.

Agrobacterium type IV secretion system and its substrates form helical arrays around the circumference of virulence-induced cells.

The genetic transformation of plant cells by Agrobacterium tumefaciens results from the transfer of DNA and proteins via a specific virulence (vir) -induced type IV secretion system (T4SS). To better understand T4SS function, we analyzed the localization of its structural components and substrates by deconvolution fluorescence microscopy. GFP fusions to T4SS proteins with cytoplasmic tails, VirB8 and VirD4, or cytoplasmic T4SS substrate proteins, VirD2, VirE2, and VirF, localize in a helical pattern of fluorescent foci around the perimeter of the bacterial cell. All fusion proteins were expressed at native levels of vir induction. Importantly, most fusion proteins are functional and do not exhibit dominant-negative effects on DNA transfer to plant cells. Further, GFP-VirB8 complements a virB8 deletion strain. We also detect native VirB8 localization as a helical array of foci by immunofluorescence microscopy. T4SS foci likely use an existing helical scaffold during their assembly. Indeed, the bacterial cytoskeletal component MinD colocalizes with GFP-VirB8. Helical arrays of foci are found at all times investigated between 12 and 48 h post vir induction at 19 degrees C. These data lead to a model with multiple T4SSs around the bacterial cell that likely facilitate host cell attachment and DNA transfer. In support, we find multiple T pili around vir-induced bacterial cells.

Intercellular trafficking of macromolecules during embryogenesis.

Plasmodesmata provide routes for communication and nutrient transfer between plant cells by interconnecting the cytoplasm of adjacent cells. A simple fluorescent tracer-loading assay was developed to monitor patterns of cell to cell transport via plasmodesmata specifically during embryogenesis. A developmental transition in plasmodesmatal size exclusion limit was found to occur at the torpedo stage of embryogenesis in Arabidopsis; at this time, plasmodesmata are downregulated, allowing transport of small (approximately 0.5 kDa) but not large (approximately 10 kDa) tracers. This assay system was used to screen for embryo defective mutants, designated increased size exclusion limit of plasmodesmata that maintain dilated plasmodesmata at the torpedo stage.

Cell-to-cell communication via plasmodesmata during Arabidopsis embryogenesis.

In Arabidopsis embryogenesis, positional information establishes the overall body plan and lineage-dependent cell fate specifies local patterning. Position-dependent gene expression and responses to the plant hormone auxin are also crucial. Recently, another mechanism that delivers positional information has been uncovered. This pathway utilizes cell-to-cell communication via plasmodesmata. Plasmodesmata span the walls between neighboring plant cells. Groups of cells that allow intercellular transport of biotic and abiotic tracers form symplastic domains of shared communication. Initially, cells of the embryo form one symplast. As development proceeds, symplastic sub-domains that correspond to the major morphological regions of the plant (i.e. shoot apex, cotyledons, hypocotyl, and root) are formed. These sub-domains further resolve into tissue-specific domains of communication (such as protodermal and vascular regions). Cell-to-cell communication via plasmodesmata between embryonic and maternal tissues ceases as development proceeds.

Plasmodesmata enable multicellularity: new insights into their evolution, biogenesis, and functions in development and immunity.

Plant cells are connected by plasmodesmata (PD), cytosolic bridges that allow molecules to freely move across the cell wall. Recently resolved relationships among land plants and their algal relatives reveal that land plants evolved PD independently from algae. Proteomic and genetic screens illuminate new dimensions of the structural and regulatory pathways that control PD biogenesis. Biochemical studies demonstrate that immunological signals induce systemic defenses by moving from diseased cells through PD; subsequently, PD transport is restricted to quarantine diseased cells. Here, we review our expanding knowledge of the roles of PD in plant development, physiology, and immunity.

Loss of PopZ At activity in Agrobacterium tumefaciens by Deletion or Depletion Leads to Multiple Growth Poles, Minicells, and Growth Defects.

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.

The cytosol must flow: intercellular transport through plasmodesmata.

Plant cells are connected across cell walls by nanoscopic channels called plasmodesmata (PD), which allow plant cells to share resources and exchange signaling molecules. Several protein components of PD membranes have been identified, and recent advances in superresolution live-cell microscopy are illuminating PD ultrastructure. Restricting transport through PD is crucial for morphogenesis, since hormones and hundreds of transcription factors regularly move through PD, and this transport must stop to allow cells to begin differentiating. Chloroplasts and mitochondria regulate PD function through signal transduction networks that coordinate plant physiology and development. Recent discoveries on the relationships of land plants and their algal relatives suggest that PD have evolved independently in several lineages, emphasizing the importance of cytosolic bridges in multicellular biology.

The essential features and modes of bacterial polar growth.

Polar growth represents a surprising departure from the canonical dispersed cell growth model. However, we know relatively little of the underlying mechanisms governing polar growth or the requisite suite of factors that direct polar growth. Underscoring how classic doctrine can be turned on its head, the peptidoglycan layer of polar-growing bacteria features unusual crosslinks and in some species the quintessential cell division proteins FtsA and FtsZ are recruited to the growing poles. Remarkably, numerous medically important pathogens utilize polar growth, accentuating the need for intensive research in this area. Here we review models of polar growth in bacteria based on recent research in the Actinomycetales and Rhizobiales, with emphasis on Mycobacterium and Agrobacterium species.

Evolution. Comment on "A promiscuous intermediate underlies the evolution of LEAFY DNA binding specificity".

Sayou et al. (Reports, 7 February 2014, p. 645) proposed a new model for evolution of transcription factors without gene duplication, using LEAFY as an archetype. Their proposal contradicts the evolutionary history of plants and ignores evidence that LEAFY evolves through gene duplications. Within their data set, we identified a moss with multiple LEAFY orthologs, which contests their model and supports that LEAFY evolves through duplications.

Peptidoglycan synthesis machinery in Agrobacterium tumefaciens during unipolar growth and cell division.

UNLABELLED: The 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. IMPORTANCE: Many 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.

Plasmodesmata dynamics are coordinated by intracellular signaling pathways.

Membrane-lined channels called plasmodesmata (PD) connect the cytoplasts of adjacent plant cells across the cell wall, permitting intercellular movement of small molecules, proteins, and RNA. Recent genetic screens for mutants with altered PD transport identified genes suggesting that chloroplasts play crucial roles in coordinating PD transport. Complementing this discovery, studies manipulating expression of PD-localized proteins imply that changes in PD transport strongly impact chloroplast biology. Ongoing efforts to find genes that control root and stomatal development reveal the critical role of PD in enforcing tissue patterning, and newly discovered PD-localized proteins show that PD influence development, intracellular signaling, and defense against pathogens. Together, these studies demonstrate that PD function and formation are tightly integrated with plant physiology.

Plasmodesmata paradigm shift: regulation from without versus within.

Plant cells are surrounded by cellulosic cell walls, creating a potential challenge to resource sharing and information exchange between individual cells. To overcome this, plants have evolved channels called plasmodesmata that provide cytoplasmic continuity between each cell and its immediate neighbors. We first review plasmodesmata basics-their architecture, their origin, the types of cargo they transport, and their molecular components. The bulk of this review discusses the regulation of plasmodesmata formation and function. Historically, plasmodesmata research has focused intensely on uncovering regulatory or structural proteins that reside within or immediately adjacent to plasmodesmata. Recent findings, however, underscore that plasmodesmata are exquisitely sensitive to signals far removed from the plasmodesmal channel itself. Signals originating from molecules and pathways that regulate cellular homeostasis-such as reactive oxygen species, organelle-organelle signaling, and organelle-nucleus signaling-lead to astonishing alterations in gene expression that affect plasmodesmata formation and function.