Patricia C. Zambryski

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.

Significance: Bacteria are ubiquitous, essential, and remarkably diverse. Many infectious species do not follow rules established in model bacteria where growth occurs by insertion of new material along the cell length; instead, many pathogens grow from their poles. Pathogenic bacteria also carry multipartite genomes for adaptation to different ecological niches. How multiple chromosomes replicate and segregate in the context of polar growth is largely unknown. Here, four replicons of the plant pathogen Agrobacterium tumefaciens exhibit distinct temporal and spatial replication/segregation patterns during the polar growth cycle, and specific proteins, targeted to the nongrowing and new growth poles, regulate replicon transmission. Uncovering the mechanisms underlying polar growth and chromosome segregation will present new opportunities to design strategies to limit bacterial diseases.

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.

Plasmodesmata (PD) are essential for plant development, but little is known about their regulation. Several studies have linked PD transport to chloroplast-centered signaling networks, but the physiological significance of this connection remains unclear. Here, we show that PD transport is strongly regulated by light and the circadian clock. Light promotes PD transport during the day, but light is not sufficient to increase rates of PD transport at night, suggesting a circadian gating mechanism. Silencing expression of the core circadian clock gene, LHY/CCA1, allows light to strongly promote PD transport during subjective night, confirming that the canonical plant circadian clock controls the PD transport light response. We conclude that PD transport is dynamically regulated during the day/night cycle. Due to the many roles of PD in plant biology, this discovery has strong implications for plant development, physiology, and pathogenesis.

Significance: The extensively studied rod-shaped bacteria Escherichia coli , Bacillus subtilis , and Caulobacter crescentus grow by dispersed insertion of new cell wall material along the entire length of the cell. An alternative prokaryotic growth mode—polar growth—is used by some Actinomycetales and Proteobacteria. The latter phylum includes the family Rhizobiaceae, of which Agrobacterium tumefaciens is a prominent member. The factors responsible for polar growth are largely unknown. Here we describe GROWTH POLE RING (GPR) protein from Agrobacterium with a striking localization as a ring of six foci at the growth pole. GPR is potentially the polar growth organizer in Agrobacterium , as its absence leads to severe morphological defects, including loss of cylindrical cell shape, while its overproduction leads to ectopic growth poles.

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 popZ _At deletion strain (Δ popZ _At ) 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 Δ popZ _At 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 _At . IMPORTANCE Agrobacterium 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.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, ...

Agrobacterium tumefaciens is a rod-shaped gram-negative bacterium, which elongates by unipolar addition of new cell envelope material. Approaching cell division, the growth pole transitions to a non-growing 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_Cc (PopZ_At) localizes specifically to growth poles. In contrast, the A. tumefaciens homolog of the C. crescentus polar organelle development protein PodJ_Cc (PodJ_At) 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 podJ_At . ΔpodJ_At cells display ectopic growth poles (branching), growth poles that fail to transition to an old pole, and elongated cells that fail to divide. In ΔpodJ_At cells, PopZ_At -GFP persists at non-transitioning growth poles post division and also localizes to ectopic growth poles, as expected for a growth pole specific factor. Even though GFP-PodJ_At does not localize to the midcell in wild type, deletion of podJ_At 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 PodJ_At is a critical factor for polar growth, and ΔpodJ_At displays a cell division phenotype, likely because the growth pole cannot transition to an old pole.