Search

DNA conjugation is a contact-dependent horizontal gene transfer mechanism responsible for disseminating drug resistance among bacterial species. Conjugation remains poorly characterised at the cellular scale, particularly regarding the reactions occurring after the plasmid enters the new host cell. Here, we use live-cell microscopy to visualise the intracellular dynamics of conjugation in real time. We reveal that the transfer of the plasmid in single-stranded DNA (ssDNA) form followed by its conversion into double-stranded DNA (dsDNA) are fast and efficient processes that occur with specific timing and subcellular localisation. Notably, the ss-to-dsDNA conversion is the critical step that governs the timing of plasmid-encoded protein production. The leading region that first enters the recipient cell carries single-stranded promoters that allow the early and transient synthesis of leading proteins immediately upon entry of the ssDNA plasmid. The subsequent ss-to-dsDNA conversion turns off leading gene expression and licences the expression of the other plasmid genes under the control of conventional double-stranded promoters. This elegant molecular strategy evolved by the conjugative plasmid allows for the timely production of factors sequentially involved in establishing, maintaining and disseminating the plasmid.

Drug-efflux by pump proteins is one of the major mechanisms of antibiotic resistance in bacteria. Here, we use quantitative fluorescence microscopy to investigate the real-time dynamics of drug accumulation and efflux in live E. coli cells. We visualize simultaneously the intrinsically fluorescent protein-synthesis inhibitor tetracycline (Tc) and the fluorescently labelled Tc-specific efflux pump, TetA. We show that Tc penetrates the cells within minutes and accumulates to stable intracellular concentration after ∼20 min. The final level of drug accumulation reflects the balance between Tc-uptake by the cells and Tc-efflux by pump proteins. In wild-type Tc-sensitive cells, drug accumulation is significantly limited by the activity of the multidrug efflux pump, AcrAB-TolC. Tc-resistance wild-type cells carrying a plasmid-borne Tn10 transposon contain variable amounts of TetA protein, produced under steady-state repression by the TetR repressor. TetA content heterogeneity determines the cells’ initial ability to efflux Tc. Yet, efflux remains partial until the synthesis of additional TetA pumps allows for Tc-efflux activity to surpass Tc-uptake. Cells overproducing TetA no longer accumulate Tc and become resistant to high concentrations of the drug. This work uncovers the dynamic balance between drug entry, protein-synthesis inhibition, efflux-pump production, drug-efflux activity and drug-resistance levels.

International audience; Auxin response factors (ARF) are key players in plant development. They mediate the cellular response to the plant hormone auxin by activating or repressing the expression of downstream developmental genes. The pivotal activation function of ARF proteins is enabled by their four-domain architecture, which includes both DNA-binding and protein dimerization motifs. To determine the evolutionary origin of this characteristic architecture, we built a comprehensive data set of 224 ARF-related protein sequences that represents all major living divisions of land plants, except hornworts. We found that ARFs are split into three subfamilies that could be traced back to the origin of the land plants. We also show that repeated events of extensive gene duplication contributed to the expansion of those three original subfamilies. Further examination of our data set uncovered a broad diversity in the structure of ARF transcripts and allowed us to identify an additional conserved motif in ARF proteins. We found that additional structural diversity in ARF proteins is mainly generated by two mechanisms: genomic truncation and alternative splicing. We propose that the loss of domains from the canonical, four-domain ARF structure has promoted functional shifts within the ARF family by disrupting either dimerization or DNA-binding capabilities. For instance, the loss of dimerization domains in some ARFs from moss and spikemoss genomes leads to proteins that are reminiscent of Aux/IAA proteins, possibly providing a clue on the evolution of these modulators of ARF function. We also assessed the functional impact of alternative splicing in the case of ARF4, for which we have identified a novel isoform in Arabidopsis thaliana. Genetic analysis showed that these two transcripts exhibit markedly different developmental roles in A. thaliana. Gene duplications, domain rearrangement, and post-transcriptional regulation have thus enabled a subtle control of auxin signaling through ARF proteins that may have contributed to the critical importance of these regulators in plant development and evolution.

Here we analyze the structural evolution of the paralogous transcription factors ETTIN (ETT/ARF3) and AUXIN RESPONSE FACTOR 4 (ARF4), which control the development of floral organs and leaves in the model angiosperm Arabidopsis. ETT is truncated at its C terminus, and consequently lacks two regulatory domains present in most other ARFs, including ARF4. Our analysis indicates ETT and ARF4 to have been generated by the duplication of a non-truncated ARF gene prior to the radiation of the extant angiosperms. We furthermore show that either ETT or ARF4 orthologs have become modified to encode truncated ARF proteins, lacking C-terminal regulatory domains, in representatives of three groups that separated early in angiosperm evolution: Amborellales, Nymphaeales and the remaining angiosperm clade. Interestingly, the production of truncated ARF4 transcripts in Amborellales occurs through an alternative splicing mechanism, rather than through a permanent truncation, as in the other groups studied. To gain insight into the potential functional significance of truncations to ETT and ARF4, we tested the capacity of native, truncated and chimeric coding sequences of these genes to restore a wild-type phenotype to Arabidopsis ett mutants. We discuss the results of this analysis in the context of the structural evolution of ARF genes in the angiosperms.

International audience; Plant and animals have evolved different strategies for their development. Whether this is linked to major differences in their cell mechanics remains unclear, mainly because measurements on plant and animal cells relied on independent experiments and setups, thus hindering any direct comparison. In this study we used the same micro-rheometer to compare animal and plant single cell rheology. We found that wall-less plant cells exhibit the same weak power law rheology as animal cells, with comparable values of elastic and loss moduli. Remarkably, microtubules primarily contributed to the rheological behavior of wall-less plant cells whereas rheology of animal cells was mainly dependent on the actin network. Thus, plant and animal cells evolved different molecular strategies to reach a comparable cytoplasmic mechanical core, suggesting that evolutionary convergence could include the internal biophysical properties of cells.

International audience; Organ morphogenesis largely relies on cell division and elongation, which need to be both coordinated between cells and orchestrated with cytoskeleton dynamics. However, components that bridge the biological signals and the effectors that define cell shape remain poorly described. We have addressed this issue through the functional characterisation of QUIRKY (QKY), previously isolated as being involved in the STRUBBELIG (SUB) genetic pathway that controls cell-cell communication and organ morphogenesis in Arabidopsis. QKY encodes a protein containing multiple C2 domains and transmembrane regions, and SUB encodes an atypical LRR-receptor-like kinase. We show that twisting of the gynoecium observed in qky results from the abnormal division pattern and anisotropic growth of clustered cells arranged sporadically along the gynoecium. Moreover, the cortical microtubule (CMT) network of these cells is disorganised. A cross to botero, a katanin mutant in which the normal orientation of CMTs and anisotropic cell expansion are impaired, strongly reduces silique deviation, reinforcing the hypothesis of a role for QKY in CMT-mediated cell growth anisotropy. We also show that QKY is localised at the plasma membrane and functions in a multiprotein complex that includes SUB and PAL OF QUIRKY (POQ), a previously uncharacterised PB1-domain-containing protein that localises both at the plasma membrane and in intracellular compartments. Our data indicate that QKY and its interactors play central roles linking together cell-cell communication and cellular growth.

Here we analyze the structural evolution of the paralogous transcription factors ETTIN (ETT/ARF3) and AUXIN RESPONSE FACTOR 4 (ARF4), which control the development of floral organs and leaves in the model angiosperm Arabidopsis. ETT is truncated at its C terminus, and consequently lacks two regulatory domains present in most other ARFs, including ARF4. Our analysis indicates ETT and ARF4 to have been generated by the duplication of a non-truncated ARF gene prior to the radiation of the extant angiosperms. We furthermore show that either ETT or ARF4 orthologs have become modified to encode truncated ARF proteins, lacking C-terminal regulatory domains, in representatives of three groups that separated early in angiosperm evolution: Amborellales, Nymphaeales and the remaining angiosperm clade. Interestingly, the production of truncated ARF4 transcripts in Amborellales occurs through an alternative splicing mechanism, rather than through a permanent truncation, as in the other groups studied. To gain insight into the potential functional significance of truncations to ETT and ARF4, we tested the capacity of native, truncated and chimeric coding sequences of these genes to restore a wild-type phenotype to Arabidopsis ett mutants. We discuss the results of this analysis in the context of the structural evolution of ARF genes in the angiosperms.

International audience

† Background and Aims CRABS CLAW (CRC) encodes a transcription factor of the YABBY family that plays important roles in carpel and nectary development in Arabidopsis thaliana. Combined evolutionary and developmental studies suggest an ancestor of the CRC gene to have controlled carpel development in the last common ancestor of the angiosperms. Roles for CRC orthologues in leaf development and carpel specification in rice, and in nectary development in core eudicots, have accordingly been interpreted as derived. The aim of this study was to assess the capacity of CRC orthologues from a basal angiosperm and from rice to complement CRC mutants of arabidopsis. These experiments were designed to test the hypothesized ancestral role of CRC in the angiosperms, and to indicate whether putatively novel roles of various CRC orthologues resulted from changes to their encoded proteins, or from other molecular evolutionary events. † Methods The crc-1 mutant of arabidopsis was genetically transformed with the coding sequences of various CRC orthologues, and with paralogous YABBY coding sequences, under the control of the arabidopsis CRC promoter. The phenotypes of transformed plants were assessed to determine the degree of complementation of the crc-1 mutant phenotype in carpel fusion, carpel form and nectary development. † Key Results The CRC orthologue from the basal angiosperm Amborella trichopoda partially complemented the crc-1 mutant phenotype in carpels, but not in nectaries. The CRC orthologue from rice partially complemented all aspects of the crc-1 mutant phenotype. Though most non-CRC YABBY coding sequences did not complement crc-1 mutant phenotypes, YABBY2 (YAB2) proved to be an exception. † Conclusions The data support a hypothesized ancestral role for CRC in carpel development and suggest that novel roles for CRC orthologues in monocots and in core eudicots resulted principally from molecular changes other than those affecting their coding sequences.

Changing shape is changing structure. Deciphering the mechanical contribution of the structural elements of the cells in shape changes is thus crucial to link the mechanical control of growth with development. Many measurements on plant and animal cells rather stress the differences in mechanical properties between both kingdoms. However, this conclusion relies on independent measurements, with very different set-ups and tissues, thus impairing any quantitative comparison. Here we took advantage of a previously described micro-rheometer to compare animal and plant single cell rheology with the same set-up. Using this method, we were able to quantitatively assess the dominant elastic behavior of plant cells in different conditions, and compare it with the viscoelastic behavior of animal cells. Surprisingly, we found that wall-less plant cells exhibit the same rheology as animal cells. This suggests that, despite the main structural differences between animal and plant cells, they also share a common mechanical core. Further investigations revealed that microtubules were the main responsible for the rheological behavior of wall-less plant cells whereas the mechanical properties of animal cells were mainly dependent on the actin network. Thus, wall-less plant cells and animal cells may have developed different strategies to converge to the same mechanical behavior.